Protein modification of living cells using sortase

ABSTRACT

Non-genetically engineered mammalian cells modified by sortase-mediated conjugation of an agent thereto are provided. Methods of conjugating agents to non-genetically engineered mammalian cells using sortase are provided. Methods of using the cells, e.g., for diagnostic and/or therapeutic purposes, are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. patent application U.S. Ser. No. 14/890,296, filedNov. 10, 2015, which is a national phase filing under 35 U.S.C. § 371 ofinternational PCT application, PCT/US2014/037545, filed May 9, 2014,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication, U.S. Ser. No. 61/822,092, filed on May 10, 2013, and toU.S. Provisional Application, U.S. Ser. No. 61/943,094, filed on Feb.21, 2014, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was made with government support under Grant No. R01A1087879 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Bacterial sortases were originally identified as enzymes that covalentlyattach proteins to the bacterial cell wall. For example, Staphylococcusaureus sortase A recognizes a set of diverse substrates via a sortaserecognition motif (e.g., LPXTG) and cleaves the peptide bond betweenthreonine and glycine, thereby releasing the residues C-terminal to thethreonine and yielding an amide linkage with the N terminus of apentaglycine nucleophile, which is provided in vivo by a cell wallprecursor.

The transpeptidation reaction catalyzed by sortases has emerged as aversatile method for site-specific modification of proteins and has beenapplied to a variety of in vitro reactions. The method has provedversatile in part because the enzyme tolerates a wide variety ofsubstrates in proximity of the cleavage site and in nucleophiles. Inmany sortase-based protein modification methods a protein to be modifiedis engineered to contain a sortase recognition motif (e.g., LPXTG) at ornear its C-terminus. When incubated with sortase and a synthetic peptidecontaining one or more N-terminal glycine residues, such artificialsortase substrates undergo a transacylation reaction resulting in theexchange of residues C-terminal to the threonine residue with thesynthetic peptide, resulting in the protein C-terminus being ligated tothe N-terminus of the synthetic peptide. In some cases, a protein to bemodified is engineered to contain one or more N-terminal glycineresidues near its N-terminus. When incubated with sortase and asynthetic peptide containing a sortase recognition motif and a sortase,the transacylation reaction results in the exchange of residuesC-terminal to the threonine residue in the synthetic peptide with themodified protein, resulting in the synthetic peptide being ligated tothe N-terminus of the protein. The synthetic peptides used in eitherapproach may be fused or conjugated to any of a number of differentmoieties. When the synthetic peptide and protein are conjugated viasortase-mediated transacylation, such moieties become attached to theprotein.

The sortase-catalyzed reaction has been used for, among other things,ligating proteins and/or peptides to one another in vitro, conjugating aprotein or peptide to a solid support or polymer, and linking a label toa protein or peptide.

SUMMARY

Some aspects of this invention relate to sortase-mediated modificationof proteins expressed by living animal cells, wherein the cells are notgenetically engineered to express a protein comprising a sortaserecognition sequence or a sequence capable of serving as a nucleophilicacceptor sequence in a sortase-mediated reaction. In some embodimentsthe animal cells are not genetically engineered. In some embodiments theanimal cells are mammalian cells, e.g., human cells. In some embodimentsthe cells are immune system cells. In some embodiments the methodsprovide for attaching any moiety of interest to a living animal cell,without requiring that the animal cell be genetically engineered andwithout requiring the use of crosslinking reagents.

Any of a wide variety of agents may be conjugated to a protein expressedby an animal cell in accordance with various embodiments. In someembodiments, a protein is modified by the conjugation of a sortasesubstrate comprising an amino acid, a peptide, a protein, apolynucleotide, a carbohydrate, a tag, a metal atom, a contrast agent, acatalyst, a non-polypeptide polymer, a recognition element, a smallmolecule, a lipid, a linker, a label, an epitope, an antigen, atherapeutic agent, a toxin, a radioisotope, a particle, or moietycomprising a reactive chemical group, e.g., a click chemistry handle.

In some embodiments, a method comprises contacting a living animal cellwith a sortase and a sortase substrate comprising a sortase recognitionmotif, wherein the animal cell is not genetically engineered to expressa protein comprising a sortase recognition sequence or a sequencecapable of serving as a nucleophilic acceptor sequence in a reactioncatalyzed by the sortase. In some embodiments the animal cell is notgenetically engineered. In some embodiments contacting is performedunder conditions suitable for the sortase to transamidate the sortasesubstrate and a polypeptide exposed at the surface of the animal cell,thereby conjugating the sortase substrate to the polypeptide. In someembodiments the sortase substrate comprises a sortase recognition motifand a moiety of interest, e.g., an amino acid, a peptide, a protein, apolynucleotide, a carbohydrate, a tag, a metal atom, a contrast agent, acatalyst, a non-polypeptide polymer, a recognition element, a smallmolecule, a lipid, a linker, a label, an epitope, an antigen, atherapeutic agent, a toxin, a radioisotope, a particle, or a clickchemistry handle. Conjugating the sortase substrate to the polypeptideexposed at the surface of animal cell attaches the moiety of interest tothe cell expressing the polypeptide.

In some embodiments a sortase substrate comprises an antibody, e.g., asingle chain antibody such as a camelid antibody, a single-domainantibody, a VHH domain, a nanobody, or an scFv. In some embodiments asortase substrate comprises a binding moiety. In some embodiments abinding moiety may comprise an antibody, polypeptide, affibody,adnectin, anticalin, or aptamer. In some embodiments a binding moietymay serve as a targeting moiety. In some embodiments a binding moietybinds to a cell surface marker of a target cell. In some embodiments atarget cell is a cancer cell, infected cell, or other abnormal ordiseased cell. In some embodiments a target cell is a normal cell.

In some embodiments a sortase substrate comprises a click chemistryhandle. Click chemistry handles are chemical moieties that provide areactive group that can partake in a click chemistry reaction. Clickchemistry reactions and suitable chemical groups for click chemistryreactions are well known to those of skill in the art, and include, butare not limited to terminal alkynes, azides, strained alkynes, dienes,dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols,and alkenes. For example, in some embodiments, an azide and an alkyneare used in a click chemistry reaction. In some embodiments a reactivegroup of first click chemistry handle attached to an animal cell via asortase-catalyzed reaction is reacted with a second reactive groupattached to a second entity, thereby conjugating the second entity tothe animal cell. The second reactive group may be a second clickchemistry handle that is compatible with the first click chemistryhandle. The entity may be, e.g., an amino acid, a peptide, a protein, apolynucleotide, a carbohydrate, a tag, a metal atom, a contrast agent, acatalyst, a non-polypeptide polymer, a recognition element, a smallmolecule, a lipid, a linker, a label, an epitope, an antigen, atherapeutic agent, a toxin, a radioisotope, a particle, or a cell. Insome embodiments the entity may be an antibody, e.g., a single chainantibody such as a camelid antibody, a single-domain antibody, a VHHdomain, a nanobody, or an scFv. In some embodiments the entity comprisesa binding moiety. In some embodiments a binding moiety may comprise anantibody, polypeptide, affibody, adnectin, anticalin, or aptamer.

Some aspects of this invention provide animal cells comprising one ormore modified endogenous, non-genetically engineered proteins comprisingan agent conjugated at or near its N-terminus. In some embodiments theagent comprises a moiety of interest, e.g., an amino acid, a peptide, aprotein, a polynucleotide, a carbohydrate, a tag, a metal atom, acontrast agent, a catalyst, a non-polypeptide polymer, a recognitionelement, a small molecule, a lipid, a linker, a label, an epitope, anantigen, a therapeutic agent, a toxin, a radioisotope, a particle, or aclick chemistry handle. In some embodiments, the modified endogenousprotein comprises an antigen-binding domain, for example, anantigen-binding domain of an antibody, e.g., a camelid antibody, asingle-domain antibody, a VHH domain, a nanobody, or an ScFv.

Some aspects of this invention comprise administering modified modifiedmammalian cells, e.g., modified human cells, to subjects, e.g., humansubjects. In some embodiments the modified mammalian cells enhance thesubject's immune response to a cancer cell, infected cell, or otherabnormal cell, or directly attack a cancer cell, infected cell, or otherabnormal cell. In some embodiments the modified mammalian cells have atherapeutic agent or detection agent conjugated to an endogenous proteinand serve to deliver the agent to the subject.

Some embodiments of this invention provide chimeric proteins, forexample, chimeric proteins that have been generated by conjugation oftwo proteins, wherein at least one of the proteins is an endogenousprotein expressed by a living animal cell. Some embodiments provideliving animal cells, e.g., mammalian cells, having one or more suchchimeric proteins attached to their surface.

Some embodiments provide modified modified endogenous mammalian proteinscomprising a sortase recognition motif (e.g., LPXTG) and a moietyattached to the sortase recognition motif. For example, a moiety may beattached directly to one of the amino acids of the sortase recognitionmotif or may be attached via a linker. In some embodiments, the modifiedendogenous mammalian protein comprises an antigen-binding domain, e.g.,an antibody or an antigen-binding antibody fragment. Exemplary, modifiedmammalian proteins provided herein may comprise, e.g., single chainantibody, camelid antibody, a VHH domain, a single-domain antibody, ananobody, an scFv, an adnectin, an affibody, an anticalin, an aptamer,or a click chemistry handle. In some embodiments the sortase recognitionmotif is positioned N-terminal with respect to the endogenouspolypeptide. In some embodiments the moiety is attached to theN-terminal amino acid of the sortase recognition motif or to an aminoacid positioned N-terminal to the N-terminal amino acid of the sortaserecognition motif.

In some aspects, the present disclosure provides a method of conjugatingan agent to an animal cell, the method comprising: contacting an animalcell with a sortase substrate that comprises a sortase recognitionsequence and an agent in the presence of a sortase under conditionssuitable for the sortase to conjugate the sortase substrate to anendogenous, non-engineered polypeptide expressed by the animal cell. Insome embodiments the sortase substrate is conjugated to an extracellularportion of an endogenous, non-engineered polypeptide expressed by thecell. In some embodiments the animal cell is a mammalian cell, e.g., ahuman cell. In some embodiments the cell is an immune system cell, e.g.,a lymphocyte (e.g., a T cell or NK cell), dendritic cell. In someembodiments the cell is a cytotoxic cell. In some embodiments the cellis a non-immortalized cell. In some embodiments the cell is a primarycell. In some embodiments the animal cell is not genetically engineeredto express a polypeptide comprising a sortase recognition sequence, asequence comprising one or more glycines, or both. In some embodimentsthe animal cell is not genetically engineered to express a polypeptidecomprising a sortase recognition sequence, a sequence comprising one ormore alanines, or both. In some embodiments the animal cell is notgenetically engineered to express a polypeptide comprising a sequencethat renders the polypeptide usable in a sortase-catalyzed reaction. Insome embodiments, the animal cell is not chemically engineered topresent a polypeptide comprising a sortase recognition sequence, asequence comprising one or more glycines, or both, on its surface. Insome embodiments, the animal cell is not chemically engineered topresent a polypeptide comprising a sortase recognition sequence, asequence comprising one or more alanines, or both, on its surface. Insome embodiments the animal cell is not chemically engineered to presentat its surface a moiety that renders the polypeptide usable in asortase-catalyzed reaction. In some embodiments the animal cell is notchemically engineered to present at its surface a polypeptide comprisinga sequence that renders the polypeptide usable in a sortase-catalyzedreaction. In some embodiments the cell is not stably or transientlytransfected or infected with a nucleic acid construct or vector encodinga protein comprising a sortase recognition sequence, nucleophilicacceptor sequence, or both. In some embodiments the cell is notgenetically engineered. In some embodiments the cell originates from asubject in need of evaluation or treatment for a disease of interest orfrom a donor who is immunocompatible with the subject. In someembodiments the cell originates from a subject in need of evaluation ortreatment for a disease characterized by the presence of abnormal orexcessive cells or pathogens in the subject's body or from a donor whois immunocompatible with the subject. In some embodiments the celloriginates from a subject in need treatment for a disease characterizedby deterioration or dysfunction of a tissue or organ, whereinregenerative medicine therapy may be useful. In some embodiments thecell originates from a subject in need of evaluation or treatment forcancer, an autoimmune disease, or an infection or from a donor who isimmunocompatible with the subject. In some embodiments the sortase is aSortase A, e.g., Staphylococcus aureus Sortase A. In some embodimentsthe sortase recognition sequence comprises LPXTG. In some embodimentsthe agent comprises an amino acid, a peptide, a protein, apolynucleotide, a carbohydrate, a tag, a metal atom, a chelating agent,a contrast agent, a catalyst, a polymer, a recognition element, a smallmolecule, a lipid, a label, an epitope, an antigen, a therapeutic agent,a cross-linker, a toxin, a radioisotope, an antibody, an antibodydomain, a click chemistry handle, a virus, a cell, or a particle. Insome embodiments the agent comprises a targeting moiety that binds to anepitope or antigen of interest. In some embodiments the targeting moietybinds to a tumor antigen or a viral, bacterial, fungal, or parasiteantigen, or a cellular marker. In some embodiments the agent comprisesone or more of the following: (a) a targeting moiety, (b) acostimulatory domain, (c) a signaling domain, (d) a receptor domain, (e)an activating domain, (f) an antigen-binding portion of an antigenreceptor; (g) an enzyme; (h) a cytolytic domain; (i) a pro-apoptoticdomain. In some embodiments the method comprises obtaining the cell oran ancestor of the animal cell from a subject in need of evaluation ortreatment for a disease of interest or from a donor who isimmunocompatible with the subject. In some embodiments the methodcomprises separating the animal cell that has the sortase substrateconjugated thereto from the sortase, unconjugated sortase substrate, orboth. In some embodiments the method comprises detecting the agentconjugated to the animal cell. In some embodiments the method comprisesadministering the animal cell having the agent conjugated thereto to asubject. In some aspects, the disclosure provides an isolated animalcell or population of isolated animal cells prepared according to any ofthe methods. In some embodiments the cell or population of cells issuitable for administration to a human subject.

In some aspects, the disclosure provides an isolated animal cellcomprising an endogenous, non-engineered polypeptide comprising asortase recognition sequence that has an agent conjugated thereto. Insome embodiments the sortase substrate is conjugated to an extracellularportion of an endogenous, non-engineered polypeptide expressed by thecell. In some embodiments the cell is a mammalian cell, e.g., a humancell. In some embodiments the cell is an immune system cell, e.g., alymphocyte (e.g., a T cell), NK cell, dendritic cell. In someembodiments the cell is a non-immortalized cell. In some embodiments thecell is a primary cell. In some embodiments the animal cell is notgenetically engineered to express a polypeptide comprising a sortaserecognition sequence, a sequence comprising one or more glycines, orboth. In some embodiments the animal cell is not genetically engineeredto express a polypeptide comprising a sortase recognition sequence, asequence comprising one or more alanines, or both. In some embodimentsthe cell is not genetically engineered. In some embodiments the cell isnot chemically engineered. In some embodiments the cell originates froma subject in need of evaluation or treatment for a disease of interestor from a donor who is immunocompatible with the subject. In someembodiments the cell originates from a subject in need of evaluation ortreatment for a disease characterized by the presence of abnormal orexcessive cells or pathogens in the subject's body or from a donor whois immunocompatible with the subject. In some embodiments the celloriginates from a subject in need of evaluation or treatment for cancer,an autoimmune disease, or an infection or from a donor who isimmunocompatible with the subject. In some embodiments the sortaserecognition sequence comprises LPXTG. In some embodiments the agentcomprises an amino acid, a peptide, a protein, a polynucleotide, acarbohydrate, a tag, a metal atom, a chelating agent, a contrast agent,a catalyst, a polymer, a recognition element, a small molecule, a lipid,a label, an epitope, an antigen, a therapeutic agent, a cross-linker, atoxin, a radioisotope, an antibody, an antibody domain, a clickchemistry handle, a virus, a cell, or a particle. In some embodimentsthe agent comprises a targeting moiety that binds to an epitope orantigen of interest. In some embodiments the agent comprises a targetingmoiety that binds to a tumor antigen or a viral, bacterial, fungal, orparasite antigen. In some embodiments the agent comprises one or more ofthe following: (a) a targeting moiety, (b) a costimulatory domain, (c) asignaling domain, (d) a receptor domain, (e) an activating domain, (f)an antigen-binding portion of an antigen receptor; (g) an enzyme; (h) acytolytic domain; (i) a pro-apoptotic domain. In some embodiments theagent is detectable by fluorescence activated cell sorting (FACS),fluorescence microscopy, Western blot, ELISA, chromatography, or massspectrometry after being conjugated to the cell.

In some aspects, the disclosure provides a method of administering anagent to a subject comprising: (a) providing the isolated animal cell orpopulation of animal cells described herein and (b) administering theisolated animal cell or population of animal cells to the subject.

In some aspects, the disclosure provides composition comprising: (i) ananimal cell comprising an endogenous, non-engineered polypeptidecomprising a sequence capable as serving as a nucleophile in asortase-mediated reaction; (ii) a sortase substrate comprising a sortaserecognition motif; and (iii) a sortase. In some embodiments the animalcell is a mammalian cell, e.g., a human cell. In some embodiments thesortase is a sortase A. In some embodiments the animal cell is an immunesystem cell, e.g., a lymphocyte (e.g., a T cell or NK cell) or dendriticcell. In some embodiments the cell is a cytotoxic cell. In someembodiments the cell is a non-immortalized cell. In some embodiments thecell is a primary cell. In some embodiments the cell is not geneticallyengineered to express a polypeptide comprising a sortase recognitionsequence, a sequence comprising one or more glycines, or both. In someembodiments the cell is not genetically engineered to express apolypeptide comprising a sortase recognition sequence, a sequencecomprising one or more alanines, or both. In some embodiments the cellis not genetically engineered. In some embodiments the cell is notchemically engineered. In some embodiments the cell originates from asubject in need of evaluation or treatment for a disease of interest orfrom a donor who is immunocompatible with the subject. In someembodiments the cell originates from a subject in need of evaluation ortreatment for a disease characterized by the presence of abnormal orexcessive cells or pathogens in the subject's body or from aimmunocompatible donor. In some embodiments the cell originates from asubject in need of evaluation or treatment for cancer, an autoimmunedisease, or an infection or from a donor who is immunocompatible withthe subject. In some embodiments the sortase is a Sortase A, e.g.,Staphylococcus aureus Sortase A. In some embodiments the sortaserecognition sequence comprises LPXTG. In some embodiments the agentcomprises an amino acid, a peptide, a protein, a polynucleotide, acarbohydrate, a tag, a metal atom, a chelating agent, a contrast agent,a catalyst, a polymer, a recognition element, a small molecule, a lipid,a label, an epitope, an antigen, a therapeutic agent, a cross-linker, atoxin, a radioisotope, an antibody, an antibody domain, a clickchemistry handle, a virus, a cell, or a particle. In some embodimentsthe agent comprises a targeting moiety that binds to an epitope orantigen of interest. In some embodiments the agent comprises a targetingmoiety that binds to a tumor antigen or a viral, bacterial, fungal, orparasite antigen. In some embodiments the agent comprises one or more ofthe following: (a) a targeting moiety, (b) a costimulatory domain, (c) asignaling domain, (d) a receptor domain, (e) an activating domain, (f)an antigen-binding portion of an antigen receptor; (g) an enzyme; (h) acytolytic domain; (i) a pro-apoptotic domain. In some embodiments thecell originates from a subject in need of evaluation or treatment for adisease of interest or from a donor who is immunocompatible with thesubject.

In some aspects, the disclosure provides a method of modulating animmune response of a subject to an entity of interest, the methodcomprising administering to the subject an animal cell that comprises anendogenous, non-engineered polypeptide comprising a sortase recognitionsequence that has an agent conjugated thereto, wherein the agentcomprises an antigen or epitope of the entity of interest or a targetingmoiety that binds to an antigen or epitope of the entity of interest. Insome embodiments the cell is a mammalian cell, e.g., a human cell. Insome embodiments the cell is an immune system cell, e.g., a lymphocyte(e.g., a T cell or NK cell) or dendritic cell. In some embodiments theentity of interest is a cancer cell, an infected cell, or a pathogen. Insome embodiments the antigen is a tumor antigen or a viral, bacterial,fungal, or parasite antigen. In some embodiments modulating an immuneresponse comprises stimulating an immune response directed towards theentity of interest. In some embodiments the entity of interest is a selfcell or structure. In some embodiments the entity of interest is anenvironmental allergen. In some embodiments modulating an immuneresponse comprises inhibiting an immune response directed towards theentity of interest. In some embodiments modulating an immune responsecomprises increasing or inducing tolerance towards the entity ofinterest. In some embodiments the agent comprises one or more of thefollowing: (a) a targeting moiety, (b) a costimulatory domain, (c) asignaling domain, (d) a receptor domain, (e) an activating domain, (f)an antigen-binding portion of an antigen receptor; (h) a cytolyticdomain; (i) a pro-apoptotic domain.

In some aspects, the disclosure provides a method of neutralizing asubstance in the body of a subject, the method comprising administeringto the subject an animal cell that comprises an endogenous,non-engineered polypeptide comprising a sortase recognition sequencethat has an agent conjugated thereto, wherein the agent binds to thesubstance. In some embodiments the substance comprises a toxin. In someembodiments the substance comprises an inflammatory cytokine. In someembodiments the agent comprises an antibody or antigen-binding fragmentthereof or a portion of a receptor that binds to the substance.

In some aspects, the disclosure provides a method of treating a subjectin need of treatment for deficiency of a protein, the method comprisingadministering to the subject an animal cell that comprises anendogenous, non-engineered polypeptide comprising a sortase recognitionsequence that has an agent conjugated thereto, wherein the agentcomprises the protein. In some embodiments the protein is an enzyme. Insome embodiments the protein is normally found in the blood.

In some aspects, the disclosure provides a method of treating a subjectin need of treatment for a disease, the method comprising administeringto the subject an animal cell that comprises an endogenous,non-engineered polypeptide comprising a sortase recognition sequencethat has an agent conjugated thereto, wherein the agent comprises atherapeutic agent effective for treating the disease. In someembodiments the therapeutic agent comprises a chemotherapy drug,anti-infective agent (e.g., antibacterial, antiviral, antifungal, orantiparasite agent), enzyme, or monoclonal antibody. In some embodimentsthe cell is a human cell. In some embodiments the cell originates fromthe subject or from an immunocompatible donor.

In some embodiments of any method comprising administering a cell to asubject, the subject is a human subject.

In some embodiments of any method comprising administering a cell to asubject, the cell is a human cell (e.g., an autologous orimmunocompatible cell) and the subject is a human subject.

In some embodiments of any method comprising administering a cell, thecell is administered into the circulatory system, e.g., intravenously.

The above summary is intended to give an overview over some aspects ofthis invention, and is not to be construed to limit the invention in anyway. Additional aspects, advantages, and embodiments of this inventionare described herein, and further embodiments will be apparent to thoseof skill in the art based on the instant disclosure. The entire contentsof all references cited in this document are hereby incorporated byreference.

The practice of certain aspects of the present invention may employconventional techniques of molecular biology, cell culture, recombinantnucleic acid (e.g., DNA) technology, immunology, transgenic biology,microbiology, nucleic acid and polypeptide synthesis, detection,manipulation, and quantification, and RNA interference that are withinthe ordinary skill of the art. See, e.g., Ausubel, F., et al., (eds.),Current Protocols in Molecular Biology, Current Protocols in Immunology,Current Protocols in Protein Science, and Current Protocols in CellBiology, all John Wiley & Sons, N.Y., edition as of December 2008 ormore recent editions; Sambrook, Russell, and Sambrook, MolecularCloning: A Laboratory Manual, ^(3rd) ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies—ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1988. Information regarding various diseases and diagnosis andcertain treatments of such diseases is found in Longo, D., et al.(eds.), Harrison's Principles of Internal Medicine, 18th Edition;McGraw-Hill Professional, 2011. Information regarding varioustherapeutic agents and human diseases is found in Brunton, L., et al.(eds.) Goodman and Gilman's The Pharmacological Basis of Therapeutics,12^(th) Ed., McGraw Hill, 2010 and/or Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 11th edition (July2009). Information regarding the immune system, immune system cells, andproteins and other molecules produced by immune system cells and/or thatplay a role in the immune system or immune response may be found instandard immunology textbooks such as Paul, W E (ed.), FundamentalImmunology, Lippincott Williams & Wilkins; 6th ed., 2008; Murphy, K,Janeway's Immunobiology, 8th ed., Garland Science, Taylor & FrancisGroup, London and New York (2012). All patents, patent applications,books, articles, documents, databases, websites, publications,references, etc., mentioned in this document are incorporated byreference in their entirety. In case of a conflict between thespecification and any of the incorporated references, the specification(including any amendments thereof), shall control. Applicants reservethe right to amend the specification based, e.g., on any of theincorporated material and/or to correct obvious errors. None of thecontent of the incorporated material shall limit the invention.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D. FIG. 1A. Schematic representation of a sortase-catalyzedtransacylation reaction in which a sortase substrate protein isconjugated to a nucleophile, with release of a portion of the substrateprotein comprising an epitope tag. FIG. 1B. Schematic representation ofsortase-catalyzed conjugation of G(n)-Probe to the C-terminus of aLPETG-tagged protein using sortase A. FIG. 1C. Schematic representationof sortase-catalyzed conjugation of Probe-LPETG to the N-terminus to aG(n)-tagged protein using sortase A. FIG. 1D. Schematic representationof sortase-catalyzed conjugation of LPETG-tagged probe or protein tonaturally exposed N-terminal glycine residues at the surface of cells.(G)n in FIGS. 1B-1D represents a sequence of one or more glycines.

FIG. 2. Immunoblot demonstrating sortase-catalyzed conjugation of biotinto non-genetically engineered mammalian cells. The blot showsbiotinylated proteins in cell lysate following incubation of mouse redcell-depleted splenocytes with a biotin-LPETG probe in the presence(right) or absence (left) of sortase.

FIG. 3. Flow cytometry analysis demonstrating sortase-catalyzedconjugation of biotin to non-genetically engineered mammalian cells.Mouse red cell-depleted splenocytes were incubated with biotin-LPETGprobe with or without sortase, washed with PBS, and incubated withphycoerythrin (PE)-conjugated strepavidin. Blue histograms (indicatedwith arrows) show PE signal gated on living cells incubated with (right)or without (left) sortase A. Black histograms (no arrow) show backgroundstaining on control splenocytes.

FIG. 4. Flow cytometry analysis demonostrating sortase-catalyzedconjugation of a GFP-specific VHH to non-genetically engineeredmammalian cells. Mouse red cell-depleted splenocytes were incubated witha GFP-specific VHH that contains a C-terminal LPETG with or withoutsortase. Cells were then washed with PBS and incubated with GFP. Bluehistograms (indicated with arrows) show GFP signal gated on living cellsincubated with (right) or without (left) sortase A. Black histograms (noarrow) show background staining on control splenocytes.

FIGS. 5A-5F. FIGS. 5A-5D. Immunoblots demonstrating sortase-catalyzedconjugation of a biotin-containing sortase substrate to non-geneticallyengineered S. cerevesiae cells (FIG. 5A), T. gondii cells (FIG. 5B),HEK-293T cells (FIG. 5C), and mouse splenocytes (FIG. 5D), as evidencedby streptavidin-based detection of protein. FIG. 5E. Histograms showingflow cytometric analysis of non-genetically engineered splenocytes thatwere sortagged with a biotin-containing sortase substrate (biotin-LPETG)and subsequently exposed to streptavidin-phycoerythrin (streptavidinPE). The rightmost peak in each histogram represents streptavidinPE-labeled splenocytes. FIG. 5F. Erythrocyte-depleted splenocytes wereincubated with 20 μM sortase A and 500 μM biotin-LPETG for the indicatedtimes. Cells were washed and incubated with streptavidin-PE and analyzedby flow cytometry. Scatter plots show the mean fluorescence intensity ofstreptavidin-PE staining for each time point, normalized to maximumstaining (120 minutes).

FIG. 6. Flow cytometry analysis demonstrating cytotoxicity of red celldepleted splenocytes from OTI Rag−/−mice (which express a T cellreceptor specific for the SIINFELK peptide) towards splenocytes thatdisplay SIINFEKL peptide at their cell surface.

FIGS. 7A-7D. Installation of VHHs on activated CD8+ T cells anddemonstration of cytotoxicity of sortase-labeled CD8+ cells towardstarget cells expressing VHH target antigen. In vitro activated CD8+ Tcells from OTI rag−/− mice were incubated for 1 hour at room temperaturewith or without 500 μM, 50 μM, or 5 μM of enhancer-LPETG or VHH7-LPETGand with or without 20 μM of sortase A, as indicated. FIG. 7A. Controlor sortagged cells were incubated with purified GFP protein. Binding ofGFP through conjugated enhancer-LPETG was analyzed by flow cytometry.FIG. 7B. Control or sortagged cells were incubated with purified GFPprotein. Amount of bound GFP was estimated by analyzing cell lysates bySDS-PAGE and Western blotting against GFP protein and comparing signalto a GFP standard (right lanes). FIGS. 7C-7D. Control (FIG. 7C) orsortagged cells (FIG. 7D) were incubated with splenocytes from WT micefor 20 hours. Histograms show the percentage of propidium iodidenegative CD4 and CD19 cells as compared to cells incubated withunmanipulated activated OTI CD8 T cells. Enhancer VHH is a VHH thatbinds to GFP. VHH7 is a VHH that binds to MHC Class II but does not bindto GFP. Following incubation, cells were washed, contacted with GFP, andsubjected to flow cytometry to detect GFP bound to the cells.

FIGS. 8A-8C. FIG. 8A. Toxoplasma gondii tachyzoites were incubated with500 μM TAMRA-LPETG and 20 μM sortase A for 15 minutes. Parasites werethen washed and incubated with human foreskin fibroblasts. Images showthe juxtaposition of bright and fluorescent fields. Black arrow:intracellular parasite. White arrow: invading parasite. Scale bar: 10micrometer. Right panels: zoomed images. FIG. 8B. Histogram showing thepercentage of sortagged Toxoplasma positive cells within CD19 negative(light gray bars) or CD19 positive (dark gray bars) splenocytepopulations after incubation of Enhancer-sortagged or VHH-7 sortaggedToxoplasma gondii with mouse splenocytes. FIG. 8C. Purified B cells fromWT or class II MHC k.o. mice were incubated together with control T.gondii or T. gondii sortagged with enhancer or VHH7 at a multiplicity ofinfection of 5. Fifteen hours after infection, cell lysis was measuredand normalized to uninfected (0%) and detergent-lysed B cells (100%).Error bars: standard deviation (n=3). **: p<0.01 at Student T-test.

FIG. 9. Kinetic analysis showing installation of two substrates:LPETG-biotin and single domain anti-GFP VHH (Enhancer) on cells as afunction of time at 37 degrees C.

FIG. 10. Installation of LPETG-biotin on intact cells, followed bystaining with streptavidin-PE. Panels show reaction with no enzyme (leftpanel), S. aureus Sortase A (middle panel) and Ca²⁺-independent sortaseheptamutant (right panel) at the indicated temperatures (bottom right).Mutations in heptamutant are shown in SEQ ID NOs: 4 and 7.

FIG. 11. Sequential installation of Enhancer first, then installation ofLPETG-biotin, demonstrating that there are remaining sites on the cellsthat can serve as nucleophile to accept LPETG on cells already modifiedwith Enhancer. Erythrocyte-depleted splenocytes were incubated with orwithout 500 μM enhancer-LPETG and 20 μM sortase A. After 60 minutes, 500μM biotin-LPETG was added to reactions where indicated for a further 15minutes. Dot plots show the binding of APC-conjugated streptavidin andGFP by sortagged cells after washing.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock,Comprehensive Organic Transformations, VCH Publishers, Inc., New York,1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition,Cambridge University Press, Cambridge, 1987.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, theterms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms (C₁₋₂₀ aliphatic). In certain embodiments, the aliphaticgroup has 1-10 carbon atoms (C₁₋₁₀ aliphatic). In certain embodiments,the aliphatic group has 1-6 carbon atoms (C₁₋₆ aliphatic). In certainembodiments, the aliphatic group has 1-5 carbon atoms (C₁₋₅ aliphatic).In certain embodiments, the aliphatic group has 1-4 carbon atoms (C₁₋₄aliphatic). In certain embodiments, the aliphatic group has 1-3 carbonatoms (C₁₋₃ aliphatic). In certain embodiments, the aliphatic group has1-2 carbon atoms (C₁₋₂ aliphatic). Aliphatic group substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms (C₁₋₂₀ alkyl). In anotherembodiment, the alkyl group employed contains 1-15 carbon atoms(C₁₋₁₅alkyl). In another embodiment, the alkyl group employed contains1-10 carbon atoms (C₁₋₁₀alkyl). In another embodiment, the alkyl groupemployed contains 1-8 carbon atoms (C₁₋₈ alkyl). In another embodiment,the alkyl group employed contains 1-6 carbon atoms (C₁₋₆ alkyl). Inanother embodiment, the alkyl group employed contains 1-5 carbon atoms(C₁₋₅alkyl). In another embodiment, the alkyl group employed contains1-4 carbon atoms (C₁₋₄alkyl). In another embodiment, the alkyl groupemployed contains 1-3 carbon atoms (C₁₋₃ alkyl). In another embodiment,the alkyl group employed contains 1-2 carbon atoms (C₁₋₂ alkyl).Examples of alkyl radicals include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substituents. Alkyl group substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety. The term “alkylene,” as used herein,refers to a biradical derived from an alkyl group, as defined herein, byremoval of two hydrogen atoms. Alkylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Alkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀ alkenyl). In some embodiments, thealkenyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅ alkenyl). In another embodiment, the alkenyl group employedcontains 2-10 carbon atoms (C₂₋₁₀ alkenyl). In still other embodiments,the alkenyl group contains 2-8 carbon atoms (C₂₋₈ alkenyl). In yet otherembodiments, the alkenyl group contains 2-6 carbons (C₂₋₆ alkenyl). Inyet other embodiments, the alkenyl group contains 2-5 carbons (C₂₋₅alkenyl). In yet other embodiments, the alkenyl group contains 2-4carbons (C₂₋₄ alkenyl). In yet other embodiments, the alkenyl groupcontains 2-3 carbons (C₂₋₃ alkenyl). In yet other embodiments, thealkenyl group contains 2 carbons (C₂alkenyl). Alkenyl groups include,for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike, which may bear one or more substituents. Alkenyl groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety. Theterm “alkenylene,” as used herein, refers to a biradical derived from analkenyl group, as defined herein, by removal of two hydrogen atoms.Alkenylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkenylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀ alkynyl). In some embodiments, thealkynyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅ alkynyl). In another embodiment, the alkynyl group employedcontains 2-10 carbon atoms (C₂₋₁₀ alkynyl). In still other embodiments,the alkynyl group contains 2-8 carbon atoms (C₂₋₈ alkynyl). In stillother embodiments, the alkynyl group contains 2-6 carbon atoms (C₂₋₆alkynyl). In still other embodiments, the alkynyl group contains 2-5carbon atoms (C₂₋₅ alkynyl). In still other embodiments, the alkynylgroup contains 2-4 carbon atoms (C₂₋₄ alkynyl). In still otherembodiments, the alkynyl group contains 2-3 carbon atoms (C₂₋₃ alkynyl).In still other embodiments, the alkynyl group contains 2 carbon atoms(C₂alkynyl). Representative alkynyl groups include, but are not limitedto, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like, which maybear one or more substituents. Alkynyl group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety. The term “alkynylene,” asused herein, refers to a biradical derived from an alkynylene group, asdefined herein, by removal of two hydrogen atoms. Alkynylene groups maybe cyclic or acyclic, branched or unbranched, substituted orunsubstituted. Alkynylene group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety.

The term “carbocyclic” or “carbocyclyl” as used herein, refers to an asused herein, refers to a cyclic aliphatic group containing 3-10 carbonring atoms (C₃₋₁₀carbocyclic). Carbocyclic group substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “heteroaliphatic,” as used herein, refers to an aliphaticmoiety, as defined herein, which includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, whichare optionally substituted with one or more functional groups, and thatfurther contains one or more heteroatoms (e.g., oxygen, sulfur,nitrogen, phosphorus, or silicon atoms) between carbon atoms. In certainembodiments, heteroaliphatic moieties are substituted by independentreplacement of one or more of the hydrogen atoms thereon with one ormore substituents. As will be appreciated by one of ordinary skill inthe art, “heteroaliphatic” is intended herein to include, but is notlimited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term“heteroaliphatic” includes the terms “heteroalkyl,” “heteroalkenyl,”“heteroalkynyl,” and the like. Furthermore, as used herein, the terms“heteroalkyl,” “heteroalkenyl,” “heteroalkynyl,” and the like encompassboth substituted and unsubstituted groups. In certain embodiments, asused herein, “heteroaliphatic” is used to indicate those heteroaliphaticgroups (cyclic, acyclic, substituted, unsubstituted, branched orunbranched) having 1-20 carbon atoms and 1-6 heteroatoms (C₁₋₂₀heteroaliphatic). In certain embodiments, the heteroaliphatic groupcontains 1-10 carbon atoms and 1-4 heteroatoms (C₁₋₁₀heteroaliphatic).In certain embodiments, the heteroaliphatic group contains 1-6 carbonatoms and 1-3 heteroatoms (C₁₋₆heteroaliphatic). In certain embodiments,the heteroaliphatic group contains 1-5 carbon atoms and 1-3 heteroatoms(C₁₋₅ heteroaliphatic). In certain embodiments, the heteroaliphaticgroup contains 1-4 carbon atoms and 1-2 heteroatoms (C₁₋₄heteroaliphatic). In certain embodiments, the heteroaliphatic groupcontains 1-3 carbon atoms and 1 heteroatom (C₁₋₃heteroaliphatic). Incertain embodiments, the heteroaliphatic group contains 1-2 carbon atomsand 1 heteroatom (C₁₋₂ heteroaliphatic). Heteroaliphatic groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “heteroalkyl,” as used herein, refers to an alkyl moiety, asdefined herein, which contain one or more heteroatoms (e.g., oxygen,sulfur, nitrogen, phosphorus, or silicon atoms) in between carbon atoms.In certain embodiments, the heteroalkyl group contains 1-20 carbon atomsand 1-6 heteroatoms (C₁₋₂₀ heteroalkyl). In certain embodiments, theheteroalkyl group contains 1-10 carbon atoms and 1-4 heteroatoms (C₁₋₁₀heteroalkyl). In certain embodiments, the heteroalkyl group contains 1-6carbon atoms and 1-3 heteroatoms (C₁₋₆ heteroalkyl). In certainembodiments, the heteroalkyl group contains 1-5 carbon atoms and 1-3heteroatoms (C₁₋₅ heteroalkyl). In certain embodiments, the heteroalkylgroup contains 1-4 carbon atoms and 1-2 heteroatoms (C₁₋₄ heteroalkyl).In certain embodiments, the heteroalkyl group contains 1-3 carbon atomsand 1 heteroatom (C₁₋₃ heteroalkyl). In certain embodiments, theheteroalkyl group contains 1-2 carbon atoms and 1 heteroatom (C₁₋₂heteroalkyl). The term “heteroalkylene,” as used herein, refers to abiradical derived from an heteroalkyl group, as defined herein, byremoval of two hydrogen atoms. Heteroalkylene groups may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted.Heteroalkylene group substituents include, but are not limited to, anyof the substituents described herein, that result in the formation of astable moiety.

The term “heteroalkenyl,” as used herein, refers to an alkenyl moiety,as defined herein, which further contains one or more heteroatoms (e.g.,oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) in betweencarbon atoms. In certain embodiments, the heteroalkenyl group contains2-20 carbon atoms and 1-6 heteroatoms (C₂₋₂₀ heteroalkenyl). In certainembodiments, the heteroalkenyl group contains 2-10 carbon atoms and 1-4heteroatoms (C₂₋₁₀ heteroalkenyl). In certain embodiments, theheteroalkenyl group contains 2-6 carbon atoms and 1-3 heteroatoms (C₂₋₆heteroalkenyl). In certain embodiments, the heteroalkenyl group contains2-5 carbon atoms and 1-3 heteroatoms (C₂₋₅ heteroalkenyl). In certainembodiments, the heteroalkenyl group contains 2-4 carbon atoms and 1-2heteroatoms (C₂₋₄ heteroalkenyl). In certain embodiments, theheteroalkenyl group contains 2-3 carbon atoms and 1 heteroatom (C₂₋₃heteroalkenyl). The term “heteroalkenylene,” as used herein, refers to abiradical derived from an heteroalkenyl group, as defined herein, byremoval of two hydrogen atoms. Heteroalkenylene groups may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted.

The term “heteroalkynyl,” as used herein, refers to an alkynyl moiety,as defined herein, which further contains one or more heteroatoms (e.g.,oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) in betweencarbon atoms. In certain embodiments, the heteroalkynyl group contains2-20 carbon atoms and 1-6 heteroatoms (C₂₋₂₀ heteroalkynyl). In certainembodiments, the heteroalkynyl group contains 2-10 carbon atoms and 1-4heteroatoms (C₂₋₁₀ heteroalkynyl). In certain embodiments, theheteroalkynyl group contains 2-6 carbon atoms and 1-3 heteroatoms (C₂₋₆heteroalkynyl). In certain embodiments, the heteroalkynyl group contains2-5 carbon atoms and 1-3 heteroatoms (C₂₋₅ heteroalkynyl). In certainembodiments, the heteroalkynyl group contains 2-4 carbon atoms and 1-2heteroatoms (C₂₋₄ heteroalkynyl). In certain embodiments, theheteroalkynyl group contains 2-3 carbon atoms and 1 heteroatom (C₂₋₃heteroalkynyl). The term “heteroalkynylene,” as used herein, refers to abiradical derived from an heteroalkynyl group, as defined herein, byremoval of two hydrogen atoms. Heteroalkynylene groups may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted.

The term “heterocyclic,” “heterocycles,” or “heterocyclyl,” as usedherein, refers to a cyclic heteroaliphatic group. A heterocyclic grouprefers to a non-aromatic, partially unsaturated or fully saturated, 3-to10-membered ring system, which includes single rings of 3 to 8 atoms insize, and bi- and tri-cyclic ring systems which may include aromaticfive- or six-membered aryl or heteroaryl groups fused to a non-aromaticring. These heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocyclic refers to a non-aromatic 5-, 6-, or7-membered ring or polycyclic group wherein at least one ring atom is aheteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), and the remaining ring atomsare carbon, the radical being joined to the rest of the molecule via anyof the ring atoms. Heterocycyl groups include, but are not limited to, abi- or tri-cyclic group, comprising fused five, six, or seven-memberedrings having between one and three heteroatoms independently selectedfrom the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ringhas 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds,and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen andsulfur heteroatoms may be optionally oxidized, (iii) the nitrogenheteroatom may optionally be quaternized, and (iv) any of the aboveheterocyclic rings may be fused to an aryl or heteroaryl ring. Exemplaryheterocycles include azacyclopropanyl, azacyclobutanyl,1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl,thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl,oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like,which may bear one or more substituents. Substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety.

The term “aryl,” as used herein, refers to an aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which all the ringatoms are carbon, and which may be substituted or unsubstituted. Incertain embodiments of the present invention, “aryl” refers to a mono,bi, or tricyclic C₄-C₂₀ aromatic ring system having one, two, or threearomatic rings which include, but are not limited to, phenyl, biphenyl,naphthyl, and the like, which may bear one or more substituents. Arylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety. Theterm “arylene,” as used herein refers to an aryl biradical derived froman aryl group, as defined herein, by removal of two hydrogen atoms.Arylene groups may be substituted or unsubstituted. Arylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.Additionally, arylene groups may be incorporated as a linker group intoan alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,or heteroalkynylene group, as defined herein.

The term “heteroaryl,” as used herein, refers to an aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which one ring atom isselected from S, O, and N; zero, one, or two ring atoms are additionalheteroatoms independently selected from S, O, and N; and the remainingring atoms are carbon, the radical being joined to the rest of themolecule via any of the ring atoms. Exemplary heteroaryls include, butare not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl,pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl,indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl,quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl,thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl,isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl,oxadiaziolyl, and the like, which may bear one or more substituents.Heteroaryl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety. The term “heteroarylene,” as used herein, refers to a biradicalderived from an heteroaryl group, as defined herein, by removal of twohydrogen atoms. Heteroarylene groups may be substituted orunsubstituted. Additionally, heteroarylene groups may be incorporated asa linker group into an alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Heteroarylene group substituents include, but are not limited to, any ofthe substituents described herein, that result in the formation of astable moiety.

The term “acyl,” as used herein, is a subset of a substituted alkylgroup, and refers to a group having the general formula —C(═O)R^(A),—C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A), —C(═O)N(R^(A))₂,—C(═S)R^(A), —C(═S)N(R^(A))₂, and —C(═S)S(R^(A)), —C(═NR^(A))R^(A),—C(═NR^(A))OR^(A), —C(═NR^(A))SR^(A), and —C(═NR^(A))N(R^(A))₂, whereinR^(A) is hydrogen; halogen; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;acyl; optionally substituted aliphatic; optionally substitutedheteroaliphatic; optionally substituted alkyl; optionally substitutedalkenyl; optionally substituted alkynyl; optionally substituted aryl,optionally substituted heteroaryl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, mono- or di-aliphaticamino, mono- ordi-heteroaliphaticamino, mono- or di-alkylamino, mono- ordi-heteroalkylamino, mono- or di-arylamino, or mono- ordi-heteroarylamino; or two R^(A) groups taken together form a 5- to6-membered heterocyclic ring. Exemplary acyl groups include aldehydes(—CHO), carboxylic acids (—CO₂H), ketones, acyl halides, esters, amides,imines, carbonates, carbamates, and ureas. Acyl substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “acylene,” as used herein, is a subset of a substitutedalkylene, substituted alkenylene, substituted alkynylene, substitutedheteroalkylene, substituted heteroalkenylene, or substitutedheteroalkynylene group, and refers to an acyl group having the generalformulae: —R⁰—(C═X¹)—R⁰—, —R⁰—X²(C═X¹)—R⁰—, or —R⁰—X²(C═X¹)X³—R⁰—, whereX¹, X², and X³ is, independently, oxygen, sulfur, or NR^(r), whereinR^(r) is hydrogen or optionally substituted aliphatic, and R⁰ is anoptionally substituted alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Exemplary acylene groups wherein R⁰ is alkylene includes—(CH₂)_(T)—O(C═O)—(CH₂)_(T)—; —(CH₂)_(T)—NR^(r)(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—O(C═NR^(r))—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—S(C═S)—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—S(C═NR^(r))—(CH₂)_(T)—;—(CH₂)_(T)—O(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—(C═S)—(CH₂)_(T)—; or—(CH₂)_(T)—S(C═O)—(CH₂)_(T)—, and the like, which may bear one or moresubstituents; and wherein each instance of T is, independently, aninteger between 0 to 20. Acylene substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety.

The term “amino,” as used herein, refers to a group of the formula(—NH₂). A “substituted amino” refers either to a mono-substituted amine(—NHR^(h)) of a disubstituted amine (—NR^(h) ₂), wherein the R^(h)substituent is any substituent as described herein that results in theformation of a stable moiety (e.g., an amino protecting group;aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted). In certain embodiments, the R^(h) substituents ofthe di-substituted amino group (—NR^(h) ₂) form a 5- to 6-memberedheterocyclic ring.

The term “hydroxy” or “hydroxyl,” as used herein, refers to a group ofthe formula (—OH). A “substituted hydroxyl” refers to a group of theformula (—OR^(i)), wherein R^(i) can be any substituent which results ina stable moiety (e.g., a hydroxyl protecting group; aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,nitro, alkylaryl, arylalkyl, and the like, each of which may or may notbe further substituted).

The term “thio” or “thiol,” as used herein, refers to a group of theformula (—SH). A “substituted thiol” refers to a group of the formula(—SR^(r)), wherein R^(r) can be any substituent that results in theformation of a stable moiety (e.g., a thiol protecting group; aliphatic,alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, cyano, nitro, alkylaryl,arylalkyl, and the like, each of which may or may not be furthersubstituted).

The term “imino,” as used herein, refers to a group of the formula(═NR^(r)), wherein W corresponds to hydrogen or any substituent asdescribed herein, that results in the formation of a stable moiety (forexample, an amino protecting group; aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino, hydroxyl,alkylaryl, arylalkyl, and the like, each of which may or may not befurther substituted).

The term “azide” or “azido,” as used herein, refers to a group of theformula (—N₃).

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine(bromo, —Br), and iodine (iodo, —I).

A “leaving group” is an art-understood term referring to a molecularfragment that departs with a pair of electrons in heterolytic bondcleavage, wherein the molecular fragment is an anion or neutralmolecule. See, for example, Smith, March's Advanced Organic Chemistry6th ed. (501-502). Exemplary leaving groups include, but are not limitedto, halo (e.g., chloro, bromo, iodo) and activated substituted hydroxylgroups, e.g., of the formula —OC(═O)SR^(aa), —OC(═O)R^(aa), —OCO₂R^(aa),—OC(═O)N(R^(bb))₂, —OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa),—OC(═NR^(bb))N(R^(bb))₂, —OS(═O)R^(aa), —OSO₂R^(aa), —OP(R^(cc))₂,—OP(R^(cc))₃, —OP(═O)₂R^(aa), —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂,—OP(═O)₂N(R^(bb))₂, or —OP(═O)(NR^(bb))₂ wherein R^(aa) is optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, or optionally substituted heteroaryl;R^(bb) is hydrogen, an amino protecting group, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, or optionally substituted heteroaryl; and R^(cc) ishydrogen, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, or optionally substitutedheteroaryl.

The term “agent,” as used herein, refers to any molecule, entity, ormoiety that can be conjugated to a sortase recognition motif. Forexample, an agent may be a protein, an amino acid, a peptide, apolynucleotide, a carbohydrate, a detectable label, a binding agent, atag, a metal atom, a contrast agent, a catalyst, a non-polypeptidepolymer, a synthetic polymer, a recognition element, a lipid, a linker,or chemical compound, such as a small molecule. In some embodiments, theagent is a binding agent, for example, a ligand or a ligand-bindingmolecule, streptavidin, biotin, an antibody or an antibody fragment. Insome embodiments, the agent cannot be genetically encoded. In some suchembodiments, the agent is a lipid, a carbohydrate, or a small molecule.Additional agents suitable for use in embodiments of the presentinvention will be apparent to the skilled artisan. The invention is notlimited in this respect.

The term “amino acid,” as used herein, includes any naturally occurringand nonnaturally occurring amino acid. Amino acids include withoutlimitation, natural alpha-amino acids such as the 20 common naturallyoccurring alpha-amino acids found in polypeptides and proteins (A, R, N,C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, also referred to asstandard amino acids), non-standard alpha-amino acids, and beta-aminoacids. There are many known non-standard, e.g., non-natural, amino acidsany of which may be included in the polypeptides or proteins describedherein. See, for example, S. Hunt, The Non-Protein Amino Acids inChemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett,Chapman and Hall, 1985 and/or Hughes, B. (ed.), Amino Acids, Peptidesand Proteins in Organic Chemistry, Volumes 1-4, Wiley-VCH (2009-2011);Blaskovich, M., Handbook on Syntheses of Amino Acids General Routes toAmino Acids, Oxford University Press, 2010. As used herein in thecontext of amino acid sequences, the term X or Xaa represents any aminoacid residue, e.g., any naturally occurring and/or any non-naturallyoccurring amino acid residue.

The term “binding agent” or “binding moiety” as used herein refers toany molecule or entity that binds another molecule or entity with highaffinity. In some embodiments, a binding agent binds its binding partnerwith high specificity. Examples of binding agents include, withoutlimitation, antibodies, antibody fragments, receptors, ligands,aptamers, and adnectins.

The term “click chemistry” refers to a chemical philosophy introduced byK. Barry Sharpless of The Scripps Research Institute, describingchemistry tailored to generate covalent bonds quickly and reliably byjoining small units comprising reactive groups together. Click chemistrydoes not refer to a specific reaction, but to a concept including, butnot limited to, reactions that mimic reactions found in nature. In someembodiments, click chemistry reactions are modular, wide in scope, givehigh chemical yields, generate inoffensive byproducts, arestereospecific, exhibit a large thermodynamic driving force>84 kJ/mol tofavor a reaction with a single reaction product, and/or can be carriedout under physiological conditions. In some embodiments, a clickchemistry reaction exhibits high atom economy, can be carried out undersimple reaction conditions, use readily available starting materials andreagents, uses no toxic solvents or use a solvent that is benign oreasily removed (preferably water), and/or provides simple productisolation by non-chromatographic methods (crystallisation ordistillation).

The term “click chemistry handle,” as used herein, refers to a reactant,or a reactive group, that can partake in a click chemistry reaction. Forexample, a strained alkyne, e.g., a cyclooctyne, is a click chemistryhandle, since it can partake in a strain-promoted cycloaddition (see,e.g., Table 1). In general, click chemistry reactions require at leasttwo molecules comprising click chemistry handles that can react witheach other. Such click chemistry handle pairs that are reactive witheach other are sometimes referred to herein as partner click chemistryhandles. For example, an azide is a partner click chemistry handle to acyclooctyne or any other alkyne. Exemplary click chemistry handlessuitable for use according to some aspects of this invention aredescribed herein, for example, in Tables 1 and 2. Other suitable clickchemistry handles are known to those of skill in the art.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof. Insome embodiments a peptide is between 3 and 60 amino acids long, e.g.,between 3 and 15, 15 and 30, 30 and 45, or 45 and 60 amino acids long.

The term “conjugated” or “conjugation” refers to an association of twomolecules, for example, two proteins or a protein and a small moleculeor other entity, with one another in a way that they are linked by adirect or indirect covalent or non-covalent interaction. In the contextof conjugation via a sortase mediated reaction, the conjugation is via acovalent bond the formation of which is catalyzed by sortase. In thecontext of conjugation via click chemistry, the conjugation is via acovalent bond formed by the reaction of two click chemistry handles. Insome embodiments, a protein is post-translationally conjugated toanother molecule, for example, a second protein, by forming a covalentbond between the protein and the other molecule after the protein hasbeen translated, and, in some embodiments, after the protein has beenisolated. In some embodiments, two molecules are conjugated directly toeach other. In some embodiments two molecules are conjugated via alinker connecting both molecules. For example, in some embodiments wheretwo proteins are conjugated to each other to form a protein fusion, thetwo proteins may be conjugated via a polypeptide linker, e.g., an aminoacid sequence connecting the C-terminus of one protein to the N-terminusof the other protein. In some embodiments, a protein N-terminus isconjugated to or near a C-terminus of a second protein generating an N—Cconjugated chimeric protein. In some embodiments, two proteins areconjugated at their respective C-termini, generating a C—C conjugatedchimeric protein. In some embodiments, two proteins are conjugated attheir respective N-termini, generating an N—N conjugated chimericprotein.

As used herein, a “detectable label” refers to a moiety that has atleast one element, isotope, or functional group incorporated into themoiety which enables detection of the molecule, e.g., a protein orpolypeptide, or other entity, to which the label is attached. Labels canbe directly attached (i.e., via a bond) or can be attached by a tether(such as, for example, an optionally substituted alkylene; an optionallysubstituted alkenylene; an optionally substituted alkynylene; anoptionally substituted heteroalkylene; an optionally substitutedheteroalkenylene; an optionally substituted heteroalkynylene; anoptionally substituted arylene; an optionally substituted heteroarylene;or an optionally substituted acylene, or any combination thereof, whichcan make up a tether). It will be appreciated that the label may beattached to or incorporated into a molecule, for example, a protein,polypeptide, or other entity, at any position.

In general, a label can fall into any one (or more) of five classes: a)a label which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁷⁶Br, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I,¹³¹I, ¹⁵³Gd, ¹⁶⁹Yb, and ¹⁸⁶Re; b) a label which contains an immunemoiety, which may be antibodies or antigens, which may be bound toenzymes (e.g., such as horseradish peroxidase); c) a label which is acolored, luminescent, phosphorescent, or fluorescent moieties (e.g.,such as the fluorescent label fluoresceinisothiocyanate (FITC); d) alabel which has one or more photo affinity moieties; and e) a labelwhich is a ligand for one or more known binding partners (e.g.,biotin-streptavidin, FK506-FKBP). In certain embodiments, a labelcomprises a radioactive isotope, preferably an isotope which emitsdetectable particles, such as β particles. In certain embodiments, thelabel comprises a fluorescent moiety. In certain embodiments, the labelis the fluorescent label fluoresceinisothiocyanate (FITC). In certainembodiments, the label comprises a ligand moiety with one or more knownbinding partners. In certain embodiments, the label comprises biotin. Insome embodiments, a label is a fluorescent polypeptide (e.g., GFP or aderivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., afirefly, Renilla, or Gaussia luciferase). It will be appreciated that,in certain embodiments, a label may react with a suitable substrate(e.g., a luciferin) to generate a detectable signal. Non-limitingexamples of fluorescent proteins include GFP and derivatives thereof,proteins comprising chromophores that emit light of different colorssuch as red, yellow, and cyan fluorescent proteins, etc. Exemplaryfluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP,mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2,EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO,mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry,mRaspberry, mKate2, mPlum, mNeptune, mTomato, T-Sapphire, mAmetrine,mKeima. See, e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescentprotein: properties, applications, and protocols (Methods of biochemicalanalysis, v. 47). Wiley-Interscience, Hoboken, N.J., 2006, and/orChudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010 for discussionof GFP and numerous other fluorescent or luminescent proteins. In someembodiments, a label comprises a dark quencher, e.g., a substance thatabsorbs excitation energy from a fluorophore and dissipates the energyas heat.

The term “adjuvant” encompasses substances that accelerate, prolong, orenhance the immune response to an antigen. In some embodiments anadjuvant serves as a lymphoid system activator that enhances the immuneresponse in a relatively non-specific manner, e g., without having anyspecific antigenic effect itself. For example, in some embodiments anadjuvant stimulates one or more components of the innate immune system.In certain embodiments an adjuvant enhances antigen-specific immuneresponses when used in combination with a specific antigen or antigens,e.g., as a component of a vaccine. Adjuvants include, but are notlimited to, aluminum salts (alum) such as aluminum hydroxide or aluminumphosphate, complete Freund's adjuvant, incomplete Freund's adjuvant,surface active substances such as lysolecithin, pluronic polyols,Amphigen, Avridine, bacterial lipopolysaccharides, 3-O-deacylatedmonophosphoryl lipid A, synthetic lipid A analogs or aminoalkylglucosamine phosphate compounds (AGP), or derivatives or analogs thereof(see, e.g., U.S. Pat. No. 6,113,918), L121/squalene, muramyl dipeptide,polyanions, peptides, saponins, oil or hydrocarbon and water emulsions,particles such as ISCOMS (immunostimulating complexes), etc. In someembodiments an adjuvant stimulates dendritic cell maturation. In someembodiments an adjuvant stimulates expression of one or morecostimulator(s), such as B7 or a B7 family member, by antigen presentingcells (APCs), e.g., dendritic cells. In some embodiments an adjuvantcomprises a CD40 agonist. In some embodiments a CD40 agonist comprisesan anti-CD40 antibody. In some embodiments a CD40 agonist comprises aCD40 ligand, such as CD40L. In some embodiments an adjuvant comprises aligand for a Toll-like receptor (TLR). In some embodiments an agent is aligand for one or more of TLRs 1-13, e.g., at least for TLR3, TLR4,and/or TLR9. In some embodiments an adjuvant comprises apathogen-derived molecular pattern (PAMP) or mimic thereof. In someembodiments an adjuvant comprises an immunostimulatory nucleic acid,e.g., a double-stranded nucleic acid, e.g., double-stranded RNA or ananalog thereof. For example, in some embodiments an adjuvant comprisespolyriboinosinic:polyribocytidylic acid (polyIC). In some embodiments anadjuvant comprises a nucleic acid comprising unmethylated nucleotides,e.g., a single-stranded CpG oligonucleotide. In some embodiments anadjuvant comprises a cationic polymer, e.g., a poly(amino acid) such aspoly-L-lysine, poly-L-arginine, or poly-L-ornithine. In some embodimentsan adjuvant comprises a nucleic acid (e.g., dsRNA, polyIC) and acationic polymer. For example, in some embodiments an adjuvant comprisespolyIC and poly-L-lysine. In some embodiments an adjuvant comprises acomplex comprising polyIC, poly-L-lysine, and carboxymethylcellulose(referred to as polyICLC). In some embodiments an adjuvant comprises aCD40 agonist and a TLR ligand. For example, in some embodiments anadjuvant comprises (i) an anti-CD40 antibody and (ii) animmunostimulatory nucleic acid and/or a cationic polymer. In someembodiments an adjuvant comprises an anti-CD40 antibody, animmunostimulatory nucleic acid, and a cationic polymer. In someembodiments an adjuvant comprises (i) an anti-CD40 antibody and (ii)poly(IC) or poly(ICLC). Exemplary adjuvants of use in variousembodiments are disclosed in, e.g., WO/2007/137427 and/or inWO/2009/086640 and/or in one or more references therein. In certainembodiments an adjuvant is pharmaceutically acceptable foradministration to a human subject. In certain embodiments an adjuvant ispharmaceutically acceptable for administration to a non-human subject,e.g., for veterinary purposes.

The term “antibody”, as used herein, refers to a glycoprotein belongingto the immunoglobulin superfamily. The terms antibody and immunoglobulinare used interchangeably. With some exceptions, mammalian antibodies aretypically made of basic structural units each with two large heavychains and two small light chains. There are several different types ofantibody heavy chains, and several different kinds of antibodies, whichare grouped into different isotypes based on which heavy chain theypossess. Five different antibody isotypes are known in mammals, IgG,IgA, IgE, IgD, and IgM, which perform different roles, and help directthe appropriate immune response for each different type of foreignobject they encounter. In some embodiments, an antibody is an IgGantibody, e.g., an antibody of the IgG1, 2, 3, or 4 human subclass.Antibodies from non-mammalian species (e.g., from birds, reptiles,amphibia) are also within the scope of the term, e.g., IgY antibodies.

Only part of an antibody is involved in the binding of the antigen, andantigen-binding antibody fragments, their preparation and use, are wellknown to those of skill in the art. As is well-known in the art, only asmall portion of an antibody molecule, the paratope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, forexample, are effectors of the complement cascade but are not involved inantigen binding. An antibody from which the pFc′ region has beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′) fragment (or F(ab′) 2 fragment), retainsboth of the antigen binding sites of an intact antibody. Similarly, anantibody from which the Fc region has been enzymatically cleaved, orwhich has been produced without the Fc region, designated an Fabfragment, retains one of the antigen binding sites of an intact antibodymolecule. Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single Fdfragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, W. R. (1986) The Experimental Foundations of ModernImmunology Wiley & Sons, Inc., New York; Roitt, I. (1991) EssentialImmunology, 7th Ed., Blackwell Scientific Publications, Oxford). In boththe heavy chain Fd fragment and the light chain of IgG immunoglobulins,there are four framework regions (FR1 through FR4) separatedrespectively by three complementarity determining regions (CDR1 throughCDR3). The CDRs, and in particular the CDR3 regions, and moreparticularly the heavy chain CDR3, are largely responsible for antibodyspecificity.

It is well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762, and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (HAMA) responseswhen administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′), Fab, Fv, and Fd fragments;antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/orlight chain CDR3 regions have been replaced by homologous human ornon-human sequences; antibodies in which the FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; antibodies in which the FR and/or CDR1 and/orCDR2 and/or light chain CDR3 regions have been replaced by homologoushuman or non-human sequences; and antibodies in which the FR and/or CDR1and/or CDR2 regions have been replaced by homologous human or non-humansequences. In some embodiments, the present invention provides forso-called single chain antibodies (e.g., scFv), (single) domainantibodies (sdAb), and other antibodies, which, in some embodiments,find use as intracellular antibodies. A single-chain variable fragment(scFv) is a protein comprising the variable regions of the heavy (VH)and light chains (VL) of an immunoglobulin, connected with a shortlinker peptide of, e.g., about 10 to about 25 amino acids. A divalent(or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs) can beengineered by linking two scFvs, e.g., by producing a single peptidechain with two VH and two VL regions, yielding tandem scFvs. Two sdAbsor an sdAb and an scFv can also be linked by producing them as singlepolypeptide chains. In some embodiments two scFv are joined in the formof a diabody. By using a linker that is too short to allow pairingbetween the two domains (VH and VL) on the same scFv chain (e.g., lessthan about 10 amino acids, e.g., about 5 amino acids) the domainsinstead pair with the complementary domains of another scFv chain andthereby create two antigen-binding sites. A bispecific agent may becreated by linking the VH and VL of two different antibodies A and B toform two different “cross-over” chains VH_(A)-VL_(B) and VH_(B)-VL_(A),whereby the chains recreate both antigen-binding sites on association(see, e.g., P., et al., Proc Natl Acad Sci USA. 1993; 90(14):6444-8).Domain antibodies, camelid and camelized antibodies and fragmentsthereof, for example, VHH domains, or nanobodies, such as thosedescribed in patents and published patent applications of Ablynx NV andDomantis are also encompassed in the term antibody. Also encompassed areVH domains obtained or derived from immunoglobulin novel (or new)antigen receptors (IgNAR) found in cartilaginous fish (e.g., sharks,skates and rays). See, e.g., WO 05/18629; Barelle, C., et al., Adv ExpMed Biol. (2009) 655:49-62, and/or the chapter by Flajnik and Dooley inAntibody Phage Display: Methods and Protocols, Methods in MolecularBiology, 2009. The term “antigen-binding antibody fragment,” as usedherein, refers to a fragment of an antibody that comprises the paratope,or a fragment of the antibody that binds to the antigen to which theantibody binds, with similar specificity and affinity as the intactantibody. Where the present disclosure refers to antibodies, thedisclosure provides embodiments pertaining to or using antigen-bindingfragments of such antibodies.

Antibodies, e.g., fully human monoclonal antibodies, may be identifiedusing phage display (or other display methods such as yeast display,ribosome display, bacterial display). Display libraries, e.g., phagedisplay libraries, are available (and/or can be generated by one ofordinary skill in the art) that can be screened to identify an antibodythat binds to an antigen of interest, e.g., using panning. See, e.g.,Sidhu, S. (ed.) Phage Display in Biotechnology and Drug Discovery (DrugDiscovery Series; CRC Press; 1^(st) ed., 2005; Aitken, R. (ed.) AntibodyPhage Display: Methods and Protocols (Methods in Molecular Biology)Humana Press; 2nd ed., 2009. In some embodiments, a monoclonal antibodyis produced using recombinant methods in suitable host cells, e.g.,prokaryotic or eukaryotic host cells. In some embodiments microbial hostcells (e.g., bacteria, fungi) are used. Nucleic acids encodingantibodies or portions thereof may be isolated and their sequencedetermined. Such nucleic acid sequences may be inserted into suitablevectors (e.g., plasmids) and, e.g., introduced into host cells forexpression. In some embodiments insect cells are used. In someembodiments mammalian cells, e.g., human cells, are used. In someembodiments, an antibody is secreted by host cells that produce it andmay be isolated, e.g., from culture medium. Methods for production andpurification of recombinant proteins are well known to those of ordinaryskill in the art. It will be understood that such methods may be appliedto produce and, optionally, purify, any protein of interest herein.

The term “chimeric antigen receptor” (CAR) refers to a polypeptidecomprising a cell activation domain fused to a binding domain, e.g., adomain comprising an antigen binding moiety. A cell that expresses achimeric antigen receptor may be referred to as a “CAR cell”. Ingeneral, the binding domain is or is located in an extracellular domainof the polypeptide, and the activation domain is located inside the cellin the cytoplasm (cytoplasmic domain). Upon binding of binding moiety toa ligand, the activation domain transmits an activation signal to theCAR cell, and the cell becomes activated as a result of signaling viathe activation domain. For example, upon binding of the antigen bindingmoiety to its cognate antigen (e.g., a protein, lipid, or othermolecule) the activation domain transmits an activation signal to theCAR cell, and the cell becomes activated as a result of signaling viathe activation domain. If the antigen is expressed by a target cell, theantigen binding moiety directs the specificity of the CAR cell to atarget cell of interest. Effector functions of the CAR cell, such ascell-mediated cytotoxicity, are directed to the target cell. In someembodiments the CAR cell is a T cell. In some embodiments the CAR cellis an NK cell. In some embodiments the binding domain, e.g., antigenbinding domain, comprises a single chain variable fragment. The bindingdomain is typically preceded by a signal peptide to direct the nascentCAR to the endoplasmic reticulum and subsequent surface expression. Ingeneral, any eukaryotic signal peptide sequence may be used. In someembodiments the signal peptide natively attached to the amino-terminalmost component of the CAR is used. It will be appreciated that thesignal peptide is cleaved and therefore absent in the mature CAR. Insome embodiments the cell activation domain comprises a biologicallyactive portion of the signaling domain of an antigen receptor such asthe T cell receptor complex (TCR-CD3 complex). For example, in someembodiments the cell activation domain comprises at least a portion ofthe cytoplasmic domain (endodomain) of the T cell receptor CD3ζ (zeta)chain (CD247) or CD3ε (epsilon) chain that is sufficient to activate Tcells. In some embodiments a CAR comprises all or substantially all ofthe cytoplasmic domain of the CD3ζ chain. In some embodiments, at leasta portion of CD3ζ comprising 1, 2, or 3 ITAM motifs is used. A CARtypically comprises a transmembrane domain (TMD) between theextracellular and cytoplasmic domains. In general, a TMD may comprise atleast a portion of a TMD found in any transmembrane protein, e.g., anyhuman transmembrane protein. In some embodiments the transmembraneprotein is a protein that spans the plasma membrane. In some embodimentsthe sequence of a TMD of a CAR comprises at least the sequence of analpha helical region of a naturally occurring transmembrane protein. Insome embodiments a TMD is derived from the alpha or beta chain of theT-cell receptor, CD28, CD3 zeta, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Insome embodiments a synthetic TMD may be used. One of ordinary skill inthe art will be aware of numerous transmembrane proteins and can readilyselect a TMD or design a synthetic TMD (see, e.g., Sharpe, H J, et al,Cell. 2010l 142(1):158-69 for discussion of features of TMDs andnumerous examples of such domains (e.g., in Table S2)). In someembodiments a CAR comprises the CD3zeta transmembrane domain andendodomain. In some embodiments a CAR comprises a spacer region one ormore amino acids long (e.g., a polypeptide linker) that links thebinding domain to the transmembrane domain. A spacer region sufficientlyflexible to allow the binding domain to orient in different directionsto facilitate antigen recognition may be selected. Exemplary spacerregions may comprise, e.g., the hinge region from an immunoglobulin,e.g., from IgG1, the C_(H)2 C_(H)3 region of an immunoglobulin, orportions of CD3. In some embodiments the cytoplasmic domain of a CARcomprises an activation domain and at least one domain that providesco-stimulatory signals. Examples of proteins containing suchco-stimulatory domains include CD28, 4-1BB, DAP10, ICOS, OX40, CD30,CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKNKG2C, B7-H3, a ligand that specifically binds with CD83, and anycombination thereof) to the cytoplasmic portion of the CAR to provideadditional signals to the T cell. In some embodiments a CAR comprisesmultiple co-stimulatory domains, which may increase potency and/orpersistence/proliferation of CAR cells that express the CAR, resultingin designs such as CD3zeta-CD28-4-1BB or CD3zeta-CD28-OX40 (wherein ineach case the name of the molecule indicates the protein in which theparticular signaling domain is found in nature). The order of suchdomains described herein is not to be considered limiting. In someembodiments the transmembrane domain of the CAR is the transmembranedomain of the most membrane proximal component of the cytoplasmic domainof the CAR. For example, if the cytoplasmic domain of the CAR comprisesCD3zeta-CD28-4-1BB, with the CD3zeta component being located closest tothe inner face of the plasma membrane (i.e., separated from the innerface of the plasma membrane by the fewest amino acids), the CARcomprises the transmembrane domain of CD3zeta. It should be understoodthat any of the domains of a CAR may be connected via a spacer ratherthan being directly fused to one another. Nucleic acid constructsencoding CARs may be introduced into cells by any suitable method, e.g.,by lentiviral or gamma-retroviral vector gene transfer or byelectroporation.

Examples of CARs, CAR cells, methods of making, culturing, manipulating,storing, and using CARs and CAR cells, reagents useful for generatingCAR cells such as nucleic acid constructs and vectors encoding CARs, aredescribed in the following publications: U.S. Pat. Pub. Nos:20040038886, 20110158957; 20120148552, 20130071414, 20130266551,20130280285; 20130287748; PCT application publications WO/2012/079000(PCT/US11/064191) and/or Finney, H M, et al., J Immunol. 2004;172(1):104-13; Kalos, M., et al., Sci. Transl. Med. 3, 95ra73 (2011);Porter, D L, et al., N Engl J Med 2011; 365:725-33. Such CARs, CARcells, methods of making, culturing, manipulating, storing, and usingCARs and CAR cells, reagents useful for generating CAR cells such asnucleic acid constructs and vectors encoding CARs, may be used incertain embodiments of the present invention. Furthermore, methods,compositions reagents, media, or devices described in any of theforegoing references may, wherever applicable, be used in embodiments ofthe present invention that pertain to CAR cells or that do not pertainto CAR cells. Such methods, reagents, media, or devices may, forexample, pertain to obtaining, culturing, maintaining, manipulating,expanding, storing, and/or administering cells. In some aspects, methodsdescribed herein of sortagging cells, wherein an agent is conjugated toan endogenous, non-genetically engineered polypeptide expressed by acell, may be applied to CAR cells described in or generated as describedin any of the afore-mentioned references. In some embodiments, sortaggedCAR cells may be used to treat a cancer that contains cells that expressan antigen to which the CAR binds or to treat an infection in whichinfected cells express an antigen to which the CAR binds. In someembodiments, any CAR that comprises an antigen binding moiety that bindsto a particular antigen or epitope may be modified by replacing theantigen binding moiety with a different antigen binding moiety thatbinds to the same antigen or epitope. In some embodiments, any CAR thatcomprises an antigen-binding moiety that binds to a particular antigenmay be modified by replacing the antigen-binding moiety with anantigen-binding moiety that binds to a different antigen, to generate aCAR that binds to such different antigen or epitope. Such modificationmay be accomplished by modifying the nucleic acid construct used toproduce the CAR, e.g., by replacing the sequence that encodes theantigen-binding moiety with a sequence that encodes a differentantigen-binding moiety. CAR T cells and CAR NK cells that express CARsthat specifically bind to various tumor antigens (e.g., CD19, CD20,etc.) have been tested in clinical trials for the treatment of humansubjects with cancer (e.g., hematologic malignancies) and shown benefitin some patients. The present invention contemplates the sortagging ofany CAR cells that have shown reasonable safety in a clinical trial.

The term “chimeric antibody,” as used herein, refers to an antibody, oran antigen-binding antibody fragment, conjugated to another molecule,for example, to a second antibody, or antigen-binding antibody fragment.Any antibody or antigen-binding antibody fragment, or antigen-bindingprotein domain can be used to generate a chimeric antibody. In someembodiments, a chimeric antibody comprises two conjugated antibodies, orantibody fragments, or one antibody conjugated to an antibody fragment,wherein the antigen-binding domains of the conjugated molecules binddifferent antigens or different epitopes of the same antigen. Suchchimeric antibodies are referred to herein as “bi-specific,” since theybind two different antigens/epitopes.

The term “costimulator” refers to a molecule that provides a stimulus(or second signal) that promotes or is required, in addition to antigen,for stimulation of naïve immune system cells, e.g., naïve T cells ornaïve B cells, and/or that contributes to sustaining or modifying theresponse. Naturally occurring costimulators include various moleculesexpressed on the surface of or secreted by APCs, which molecules bind toreceptors on the surfaces of, e.g., T cells. Examples of receptors towhich costimulators bind include, e.g., CD28 family members (e.g., CD28and inducible costimulator (ICOS)) and CD2 family members (e.g., CD2,SLAM). Examples of costimulators include various members of the B7family of molecules such as B7-1 and B7-2 (which bind to CD28) and ICOSligand (which binds to ICOS). In some embodiments a costimulator is aTNF alpha family member. For example, CD70 on DCs binding to itsreceptor CD27 on naïve T cells delivers costimulatory signals; 4-1BBL(also called CD137L) on APCs delivers costimulatory signals by bindingto its receptor CD137 on T cells. It will be appreciated that theeffects of an interaction may be bidirectional, e.g., APCs may receivecostimulation via their interaction with cells that they stimulate. OX40(CD134) is a secondary costimulatory molecule, expressed after typicallyabout 24 to 72 hours following activation; its ligand, OX40L, isexpressed on APCs following their activation. In some embodimentsexpression of costimulator(s) by APCs is stimulated by an adjuvant,e.g., a CD40 ligand, PAMP or PAMP mimic, or TLR ligand. In someembodiments a costimulator is a soluble molecule. In some embodiments asoluble costimulator is a recombinantly produced polypeptide comprisingat least a functional portion of the extracellular domain of a naturallyoccurring costimulator or a functional variant thereof.

The term “endogenous polypeptide” refers to a naturally occurringpolypeptide that originates naturally from or is naturally produced by acell, e.g., a polypeptide that is an expression product of a gene thatboth (i) is present in the genetic material (nuclear or mitochondrialgenome) of the cell (an “endogenous gene”) and (ii) has not beenmodified or introduced into the cell or an ancestor of the cell by thehand of man or by a virus or other vector. It will be understood thatendogenous genes of a particular species (e.g., humans) may includesequences introduced by retroviruses, transposons, or other vectors butthat have been present in the genome of at least some members of thespecies for sufficiently long to be considered endogenous. For purposeshereof, genetic elements that can be shown to have been present in thegenome of at least some individuals of a particular species, e.g., at aparticular chromosomal location, for at least 1000 years are consideredendogenous to that species. One of ordinary skill in the art will beaware of endogenous genes and polypeptides of animal cells, e.g.,mammalian cells, e.g., human cells. For purposes hereof, “introducing” anucleic acid into a cell encompasses introducing the nucleic acid itselfor introducing a nucleic acid that can undergo one or more rounds ofcopying, reverse transcription, and/or processing in the cell to yieldthe nucleic acid. An endogenous polypeptide may be processed or modifiedduring or after its synthesis. For example an N-terminal amino acid orsecretion signal sequence may be removed or a loop may be cleaved. Incertain embodiments of any aspect of the disclosure, “endogenouspolypeptides” are also not chemically modified as defined below.

The terms “genetically engineered” or “genetically modified”, or“recombinant” encompass nucleic acids whose sequence comprises anon-naturally occurring sequence, (a sequence invented or generated byman and not occurring in nature or not known to occur in nature),comprises two or more naturally occurring sequences joined together thatare not found joined to one another in their naturally occurring state,or comprises a deletion, insertion, rearrangement, or other alterationof or in a naturally occurring sequence, wherein the deletion,insertion, rearrangement, or other alteration is brought about by thehand of man. The terms “genetically engineered”, “genetically modified”,or “recombinant” polypeptide encompass polypeptides encoded bygenetically engineered nucleic acids. In some embodiments the sequenceof a genetically engineered polypeptide expressed by a cell is distinctfrom those polypeptides that are endogenous to the cell. The terms“genetically engineered cell”, “genetically modified cell”, or“recombinant” cell” encompass cells into which a nucleic acid has beenintroduced by the hand of man and their descendants that inherit atleast a portion of the introduced nucleic acid. In some embodiments agenetically engineered cell has had its genome altered by the hand ofman, e.g., by insertion of an exogenous nucleic acid sequence and/ordeletion of an endogenous nucleic acid sequence, or is descended fromsuch a cell and has inherited a copy of at least a portion of theoriginal alteration. In some embodiments the nucleic acid or a portionthereof, or a copy of the nucleic acid or a portion thereof, may beintegrated into the genome of the cell. “Non-genetically engineered,“non-genetically modified”, and “non-recombinant” refer to not beinggenetically engineered, absence of genetic modification, etc.Non-genetically engineered polypeptides encompass endogenouspolypeptides. In certain embodiments a non-genetically engineered cell,gene, or genome does not contain non-endogenous nucleic acid, e.g., DNAor RNA that originates from a vector, from a different species, or thatcomprises an artificial sequence, e.g., DNA or RNA that was introducedby the hand of man. In certain embodiments a non-genetically engineeredcell has not been intentionally contacted with a nucleic acid that iscapable of causing a heritable genetic alteration under conditionssuitable for uptake of the nucleic acid by the cells.

The terms “chemically engineered” or “chemically modified” encompassmodifications made to endogenous proteins or cells to introduce a“linker” as described below, to an endogenous protein or cell. Chemicalmodifications can be any known in the art. Examples of suchmodifications are provided in Ta et al. Circ. Res. 2011; 109: 365-373and International Publication No. WO 2012/142659, both incorporated byreference herein in their entireties. Other chemical modifications thatcan be made to biomolecules, e.g., polypeptides, are known to those ofordinary skill in the art. See, e.g., Hermanson, G., BioconjugateTechniques, Academic Press; 2nd edition (2008). In certain embodiments,chemical modification of cells includes introducing a reactivefunctional group, such as a sulfhydryl or maleimide, to cell surfaces,e.g., by attachment (e.g., via a covalent bond) to an extracellulardomain of an endogenous polypeptide, followed by labelling the cellswith a moiety capable of serving as a nucleophile in a sortase-catalyzedreaction, such as a (G)_(n)- or (A)_(n)-containing peptide, via reactionwith such reactive functional group. In certain embodiments, chemicalmodification of cells includes introducing sulfhydryls to cell surfacesvia reaction with primary amines using, e.g., 2-Iminothiolane or Traut'sreagent followed by labelling cells with NH₂GGG-tags via specificreaction of sulfhydryls on the cell surface and maleimide groups onNH₂-GGG-maleimide peptides.

The term “immunomodulator” refers to substances that are capable eitherby themselves or together with other agent(s) of inducing, enhancing,suppressing, or regulating an immune response. (It will be understoodthat the term generally does not refer to those entities that are thetarget of the immune response such as pathogens, tumor cells, grafts, orself antigens in the case of autoimmune disease). Immunomodulatorsinclude substances capable of modulating the activation, proliferation,differentiation, and/or biological activity of immune system cells.Examples, include, e.g., cytokines, costimulators, adjuvants.

The term “linker” as used herein, refers to a chemical group or moleculecovalently linked to a molecule, for example, a protein, and a chemicalgroup or moiety. In some embodiments, the linker is positioned between,or flanked by, two groups, molecules, or moieties and connected to eachone via a covalent bond, thus connecting the two. In some embodiments,the linker is an amino acid or a plurality of amino acids. In someembodiments, the linker is an organic molecule, group, or chemicalmoiety. In some embodiments a linker connects two or more polypeptides.In some embodiments a linker comprises or consists of a polypeptide. Insome embodiments a linker may comprise or consist of one or more glycineresidues and, in some embodiments, one or more serine, and/or threonineresidues. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 aminoacids. In some embodiments, the linker comprises an oligoglycinesequence. In some embodiments a linker may comprise at least 50% glycineresidues and, in some embodiments, between 5% and 50% of the amino acidsare serine or threonine (i.e., S+T is between 5% and 50%, where S is thepercentage of serine residues and T is the percentage of threonineresidues). Examples of linkers include, e.g., (Gly-Ser)_(n);(Gly-Gly-Ser)_(n); (Gly-Gly-Gly-Ser)_(n); (Gly-Gly-Gly-Gly-Ser)_(n),where n is a number sufficient to produce a desired linker length, e.g.,about 5-15 amino acids, e.g., up to about 25-50 amino acids. Theafore-mentioned sequences can be permuted and/or concatenated in anyorder and/or may be truncated and/or any of the Ser residues may bereplaced by Thr and/or any of the Gly or Ser residues may be replaced byAla. In some embodiments a linker comprises an aliphatic, alicyclic,heteroaliphatic, heteroalicyclic, aromatic, or heteroaromatic linkerwhich, in some embodiments, comprises between 1 and 6, 6 and 12, or12-30 carbon atoms in the main chain connecting the moieties at eachend. In some embodiments a linker comprises a linear saturated orunsaturated hydrocarbon chain, an oligo(ethylene glycol) chain, one ormore amino acids (e.g., a peptide), an alicyclic structure, or anaromatic ring. In some embodiments a linker is sufficiently long andflexible so as to permit linked polypeptides to assemble to form aproper three dimensional structure (e.g., as found in nature) and/orretain one or more activities such as binding, enzymatic activity,and/or appropriate interaction with its typical interaction partners. Insome embodiments a linker may comprise a protease recognition site orlabile bond, which allows release of one or both of the linked moietiesunder appropriate conditions, e.g., in the presence of a protease thatrecognizes the protease recognition site and cleaves within or near thelinker.

The term “marker” or “cellular marker” refers to any molecular moiety(e.g., protein, peptide, carbohydrate, polysaccharide, nucleic acid(mRNA or other RNA species, DNA), lipid, or a combination thereof) thatcharacterizes, indicates, or identifies one or more cell type(s), tissuetype(s), cell lineages, or embryological tissue of origin and/or thatcharacterizes, indicates, or identifies a particular physiological orpathological state, e.g., an activation state, cell cycle state,metabolic state, differentiation state, apoptotic state, diseased state,etc. In some embodiments, the presence, absence, or amount of certainmarker(s) may indicate a particular physiological or diseased state of asubject, organ, tissue, or cell. In some embodiments a cell surfacemarker is a “cluster of differentiation” (CD) molecule. Numerous CDmolecules are known in the art. See, e.g., H. Zola, et al., Leukocyteand Stromal Cell Molecules: the CD Markers, Wiley, New Jersey, 2007and/or databases cited therein; Proceedings of the 9th InternationalWorkshop on Human Leukocyte Differentiation Antigens published inImmunology Letters, Volume 134, Issue 2, Pages 103-188 (30 Jan. 2011);Human Cell Differentiation Molecules database available athttp://www.hcdm.org/MoleculeInformation/tabid/54/Default.aspx; and/orHuman and Mouse CD Handbook, available athttp://www.bdbiosciences.com/documents/cd_marker_handbook.pdf (BDBiosciences, San Jose, Calif., 2010). In some embodiments a cellularmarker is cell type specific. For example, a cell type specific markeris typically present at a higher level on or in a particular cell typeor cell types of interest than on or in many other cell types. In someinstances a cell type specific marker is present at detectable levelsonly on or in a particular cell type of interest. However, it will beappreciated that useful cell type specific markers need not beabsolutely specific for the cell type of interest. In some embodiments acell type specific marker for a particular cell type is expressed atlevels at least 3 fold greater in that cell type than in a referencepopulation of cells which may consist, for example, of a mixturecontaining cells from a plurality (e.g., 5-10 or more) of differenttissues or organs in approximately equal amounts. In some embodiments acell type specific marker is present at levels at least 4-5 fold,between 5-10 fold, or more than 10-fold greater than its averageexpression in a reference population. In some embodiments detection ormeasurement of a cell type specific marker can distinguish the cell typeor types of interest from cells of many, most, or all other types. Ingeneral, the presence and/or abundance of most markers may be determinedusing standard techniques such as Northern blotting, in situhybridization, RT-PCR, sequencing, immunological methods such asimmunoblotting, immunodetection, or fluorescence detection followingstaining with fluorescently labeled antibodies, oligonucleotide or cDNAmicroarray or membrane array, protein microarray analysis, massspectrometry, etc.

The term “naturally occurring” as applied to an entity (e.g., amolecule, substance, etc.) refers to the fact that the entity can befound in nature as distinct from being artificially created or modifiedby man. For example, a polypeptide or polynucleotide sequence that isnaturally present in a virus or in a prokaryotic (bacteria) oreukaryotic (e.g., fungal, protozoa, insect, plant, vertebrate) cell,tissue, or organism and/or that can be isolated from a source in natureand which has not been intentionally modified by man (e.g., in thelaboratory) is naturally occurring. “Non-naturally occurring” (alsoreferred to as “synthetic” or “artificial”) as applied to an entitymeans that the entity is not naturally occurring, i.e., it cannot befound in nature as distinct from being artificially produced by man. Itwill be appreciated that a “naturally occurring” entity may be producedby man, e.g., through recombinant nucleic acid techniques or chemicalsynthesis and/or may be isolated or purified. Such an entity is stillconsidered naturally occurring so long as it does not otherwise differmaterially from the entity as found in nature.

The term “purified” refers to agents that have been separated from some,many, or most of the components with which they are associated in natureor when originally generated. In general, such purification involvesaction of the hand of man. In some embodiments a purified agent is, forexample, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more than 99% pure. In some embodiments, a nucleic acid,polypeptide, or small molecule is purified such that it constitutes atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%,or more, of the total nucleic acid, polypeptide, or small moleculematerial, respectively, present in a preparation. In some embodiments,an organic substance, e.g., a nucleic acid, polypeptide, or smallmolecule, is purified such that it constitutes at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or more, of thetotal organic material present in a preparation. Purity may be based on,e.g., dry weight, size of peaks on a chromatography tracing (GC, HPLC,etc.), molecular abundance, electrophoretic methods, intensity of bandson a gel, spectroscopic data (e.g., NMR), elemental analysis, highthroughput sequencing, mass spectrometry, or any art-acceptedquantification method. In some embodiments, water, buffer substances,ions, and/or small molecules (e.g., synthetic precursors such asnucleotides or amino acids), can optionally be present in a purifiedpreparation. A purified agent may be prepared by separating it fromother substances (e.g., other cellular materials), or by producing it insuch a manner to achieve a desired degree of purity. In some embodiments“partially purified” or “at least partially purified” with respect to amolecule produced by a cell means that a molecule produced by a cell isno longer present within the cell, e.g., the cell has been lysed and,optionally, at least some of the cellular material (e.g., cell wall,cell membrane(s), cell organelle(s)) has been removed and/or themolecule has been separated or segregated from at least some moleculesof the same type (protein, RNA, DNA, etc.) that were present in thelysate or, in the case of a molecule that is secreted by a cell, themolecule has been separated from at least some components of the mediumor environment into which it was secreted. In some embodiments, anyagent disclosed herein is purified. In some embodiments a compositioncomprises one or more purified agents.

The term “RNA interference” (RNAi) encompasses processes in which amolecular complex known as an RNA-induced silencing complex (RISC)silences or “knocks down” gene expression in a sequence-specific mannerin, e.g., eukaryotic cells, e.g., vertebrate cells, or in an appropriatein vitro system. RISC may incorporate a short nucleic acid strand (e.g.,about 16-about 30 nucleotides (nt) in length) that pairs with anddirects or “guides” sequence-specific degradation or translationalrepression of RNA (e.g., mRNA) to which the strand has complementarity.The short nucleic acid strand may be referred to as a “guide strand” or“antisense strand”. An RNA strand to which the guide strand hascomplementarity may be referred to as a “target RNA”. Thecomplementarity of the structure formed by hybridization of a target RNAand the guide strand may be such that the strand can (i) guide cleavageof the target RNA in the RNA-induced silencing complex (RISC) and/or(ii) cause translational repression of the target RNA. Reduction ofexpression due to RNAi may be essentially complete (e.g., the amount ofa gene product is reduced to background levels) or may be less thancomplete in various embodiments. For example, mRNA and/or protein levelmay be reduced by 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more, invarious embodiments. As known in the art, the complementarity betweenthe guide strand and a target RNA need not be perfect (100%) but needonly be sufficient to result in inhibition of gene expression. Forexample, in some embodiments 1, 2, 3, 4, 5, or more nucleotides of aguide strand may not be matched to a target RNA. In some embodiments aguide strand has at least about 80%, 85%, or 90%, e.g., least about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarityto a target RNA over a continuous stretch of at least about 15 nt, e.g.,between 15 nt and 30 nt, between 17 nt and 29 nt, between 18 nt and 25nt, between 19 nt and 23 nt, of the target RNA. In some embodiments atleast the seed region of a guide strand (the nucleotides in positions2-7 or 2-8 of the guide strand) is perfectly complementary to a targetRNA. As used herein, the term “RNAi agent” encompasses nucleic acidsthat can be used to achieve RNAi in eukaryotic cells. Short interferingRNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) areexamples of RNAi agents. siRNAs typically comprise two separate nucleicacid strands that are hybridized to each other to form a structure thatcontains a double stranded (duplex) portion at least 15 nt in length,e.g., about 15-about 30 nt long, e.g., between 17-27 nt long, e.g.,between 18-25 nt long, e.g., between 19-23 nt long, e.g., 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Insome embodiments the strands of an siRNA are perfectly complementary toeach other within the duplex portion. In some embodiments the duplexportion may contain one or more unmatched nucleotides, e.g., one or moremismatched (non-complementary) nucleotide pairs or bulged nucleotides.In some embodiments either or both strands of an siRNA may contain up toabout 1, 2, 3, or 4 unmatched nucleotides within the duplex portion. Insome embodiments a strand may have a length of between 15-35 nt, e.g.,between 17-29 nt, e.g., 19-25 nt, e.g., 21-23 nt. Strands may be equalin length or may have different lengths in various embodiments. In someembodiments strands may differ by between 1-10 nt in length. A strandmay have a 5′ phosphate group and/or a 3′ hydroxyl (—OH) group. Eitheror both strands of an siRNA may comprise a 3′ overhang of, e.g., about1-10 nt (e.g., 1-5 nt, e.g., 2 nt). shRNAs are nucleic acid moleculesthat comprise a stem-loop structure and a length typically between about40-150 nt, e.g., about 50-100 nt, e.g., 60-80 nt. A “stem-loopstructure” (also referred to as a “hairpin” structure) refers to anucleic acid having a secondary structure that includes a region ofnucleotides which are known or predicted to form a double strand (stemportion; duplex) that is linked on one side by a region of (usually)predominantly single-stranded nucleotides (loop portion). Suchstructures are well known in the art and the term is used consistentlywith its meaning in the art. A guide strand sequence may be positionedin either arm of the stem, i.e., 5′ with respect to the loop or 3′ withrespect to the loop in various embodiments. As is known in the art, thestem structure does not require exact base-pairing (perfectcomplementarity). Thus, the stem may include one or more unmatchedresidues or the base-pairing may be exact, i.e., it may not include anymismatches or bulges. In some embodiments the stem is between 15-30 nt,e.g., between 17-29 nt, e.g., 19-25 nt. In some embodiments the stem isbetween 15-19 nt. In some embodiments the stem is between 19-30 nt. Insome embodiments the loop is between 1 and 20 nt in length, e.g., 1-15nt, e.g., 4-9 nt. The shRNA structure may comprise a 5′ or 3′ overhang.As known in the art, an shRNA may undergo intracellular processing toremove the loop and generate an siRNA. Mature endogenous miRNAs areshort (typically 18-24 nt, e.g., about 22 nt), single-stranded RNAs thatare generated by intracellular processing from larger, endogenouslyencoded precursor RNA molecules termed miRNA precursors (see, e.g.,Bartel, D., Cell. 116(2):281-97 (2004); Bartel D P. Cell. 136(2):215-33(2009); Winter, J., et al., Nature Cell Biology 11: 228-234 (2009).Artificial miRNA may be designed to take advantage of the endogenousRNAi pathway in order to silence a target RNA of interest. An RNAi agentthat contains a strand sufficiently complementary to an RNA of interestso as to result in reduced expression of the RNA of interest (e.g., as aresult of degradation or repression of translation of the RNA) in a cellor in an in vitro system capable of mediating RNAi and/or that comprisesa sequence that is at least 80%, 90%, 95%, or more (e.g., 100%)complementary to a sequence comprising at least 10, 12, 15, 17, or 19consecutive nucleotides of an RNA of interest may be referred to asbeing “targeted to” the RNA of interest. An RNAi agent targeted to anRNA transcript may also considered to be targeted to a gene from whichthe transcript is transcribed. In some embodiments an RNAi agent is avector (e.g., an expression vector) suitable for causing intracellularexpression of one or more transcripts that give rise to a siRNA, shRNA,or miRNA in the cell. Such a vector may be referred to as an “RNAivector”. An RNAi vector may comprise a template that, when transcribed,yields transcripts that may form a siRNA (e.g., as two separate strandsthat hybridize to each other), shRNA, or miRNA precursor (e.g.,pri-miRNA or pre-mRNA).

The term “sortagging,” as used herein, refers to the process ofattaching (conjugating) a tag to a target entity, e.g., a molecule, forexample, a protein, using a sortase. The term “sortagging” encompassesattaching a tag to a protein expressed by a living cell using a sortase,thereby attaching the tag to the cell. The term “tag” is used in a broadsense to encompass any of a wide variety of entities. Examples ofsuitable tags include, but are not limited to, amino acids, peptides,proteins, nucleic acids, polynucleotides, sugars, carbohydrates,polymers, lipids, fatty acids, and small molecules. Other suitable tagswill be apparent to those of skill in the art and the invention is notlimited in this aspect. In some embodiments a tag is covalently ornoncovalently attached to, physically associated with, or part ofanother entity, such as a virus, cell, particle, or other supramolecularcomplex, and attaching the tag to the target entity (sortagging thetarget entity) attaches the entity to the target entity. In someembodiments, a tag comprises a sequence useful for purifying,expressing, solubilizing, and/or detecting a polypeptide. In someembodiments, a tag may comprise two or more moieties, which may beconjugated to each other. In some embodiments a tag may serve multiplefunctions. In some embodiments a tag is a relatively small polypeptide,e.g., ranging from a few amino acids up to about 100 amino acids long.In some embodiments a tag is more than 100 amino acids long, e.g., up toabout 500 amino acids long, or more. In some embodiments, a tagcomprises an HA, TAP, Myc, 6×His, Flag, V5, or GST tag, to name fewexamples. A tag (e.g., any of the afore-mentioned tags) that comprisesan epitope against which an antibody, e.g., a monoclonal antibody, isavailable (e.g., commercially available) or known in the art may bereferred to as an “epitope tag”. In some embodiments a tag comprises asolubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, a Streptag, or a monomeric mutant of the Ocr protein of bacteriophage T7). See,e.g., Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8(2006). In some embodiments, a tag is cleavable, so that at least aportion of it can be removed, e.g., by a protease. In some embodiments,this is achieved by including a protease cleavage site in the tag, e.g.,adjacent or linked to a functional portion of the tag. Exemplaryproteases include, e.g., thrombin, TEV protease, Factor Xa, PreScissionprotease, etc. In some embodiments, a “self-cleaving” tag is used. See,e.g., PCT/US05/05763. In some embodiments, sortagging involves couplinga tag to an endogenous protein on the surface of a cell.

The term “sortase” refers to an enzyme that has transamidase activity.Sortases, also referred to as transamidases, can form a peptide linkage(i.e., amide linkage) between an appropriate acyl donor compound and anucleophilic acyl acceptor containing a NH₂—CH₂-moiety, such as anN-terminal glycine. Sortases recognize substrates comprising a sortaserecognition motif, e.g., the amino acid sequence LPXTG. A moleculerecognized by a sortase (i.e., comprising a sortase recognition motif)is sometimes termed a “sortase substrate” herein. After recognition ofsuch a motif by sortase the catalytic residue (e.g., cysteine) in theenzyme's active site serves as a nucleophile to cleave a peptide bond inthe motif (e.g., the peptide bond between threonine and glycine inLXPTG). Cleavage occurs with concomitant formation of a thioacylintermediate between substrate and enzyme. This intermediate is resolvedby reaction with an appropriate nucleophile, thereby creating a new bondthat links the substrate to the nucleophile. Sortases tolerate a widevariety of moieties in proximity to the cleavage site, thus allowing forthe versatile conjugation of diverse entities so long as the substratecontains a suitably exposed sortase recognition motif and a suitablenucleophile is available. The terms “sortase-mediated transacylationreaction”, “sortase-catalyzed transacylation reaction”,“sortase-mediated reaction”, “sortase-catalyzed reaction”, “sortasereaction” and like terms, are used interchangeably herein to refer tosuch a reaction. The terms “sortase recognition motif”, “sortaserecognition sequence”, and “transamidase recognition sequence”(sometimes abbreviated as “TRS”) with respect to sequences recognized bya transamidase or sortase, are used interchangeably herein. The term“nucleophilic acceptor sequence” refers to an amino acid sequencecapable of serving as a nucleophile in a sortase-catalyzed reaction,e.g., a sequence comprising an N-terminal glycine (e.g., 1, 2, 3, 4, or5 N-terminal glycines) or in some embodiments comprising an N-terminalalanine (e.g., 1, 2, 3, 4, or 5 N-terminal alanines).

Substrates suitable for sortase-mediated conjugation can readily bedesigned. For example, polypeptides can be modified to include a sortaserecognition motif at or near their C-terminus, thereby allowing them toserve as substrates for sortase. The sortase recognition motif need notbe positioned at the very C-terminus of a substrate but should typicallybe sufficiently accessible by the enzyme to participate in the sortasereaction. In some embodiments a sortase recognition motif is consideredto be “near” a C-terminus if there are no more than 5, 6, 7, 8, 9, 10,12, 15, 20, or 25 amino acids between the most N-terminal amino acid inthe sortase recognition motif (e.g., L) and the C-terminal amino acid ofthe polypeptide. In some embodiments there is at least 1, 2, 3, or 4additional amino acids C-terminal to a G or A in a sortase recognitionmotif. In some embodiments at least one additional amino acid is G or A.For example, the sortase substrate may comprise LPXTGG, e.g., LPETGG. Insome embodiments a tag (e.g., a 6×His tag or other small peptide tag) islocated C-terminal to the sortase recognition motif, optionallyseparated from it by a spacer. Upon cleavage, the tag is released. Thefree tag may be detected, which may be useful to monitor the progress orextent of the reaction. In some embodiments a sortase recognition motifis located in a flexible loop of a polypeptide. The flexible loop may beexposed at the surface of a properly folded polypeptide. The loop may becleaved by a protease so as to position the sortase recognition motif ator near the C-terminus of a resulting cleavage product. A polypeptidecomprising a sortase recognition motif may be modified by incorporatingor attaching any of a wide variety of moieties thereto. The resultingmodified polypeptide can serve as a sortase substrate, resulting inconjugation of the moiety to the nucleophile. Suitable nucleophiles thatcan be used in a sortase reaction typically comprise a short run (e.g.,1-10) of glycine residues, although even an alkylamine suffices to allowthe reaction to proceed. Polypeptides can be modified to comprise anucleophilic acceptor sequence, e.g., a sequence comprising one or moreglycines, at their N-terminus and the resulting polypeptide may be usedas a nucleophile in a sortase-catalyzed reaction. Such a reaction canresult in installation of any of a wide variety of entities (comprisinga sortase sortase recognition motif) at the N-terminus of thepolypeptide.

A “subject” may be any vertebrate organism in various embodiments. Asubject may be individual to whom an agent, cell, substance, orcomposition is administered, e.g., for experimental, diagnostic, and/ortherapeutic purposes or from whom a sample is obtained or on whom aprocedure is performed. In some embodiments a subject is a mammal, e.g.a human, non-human primate, rodent (e.g., mouse, rat, rabbit), ungulate(e.g., ovine, bovine, equine, caprine species), canine, or feline. Insome embodiments a subject is an avian. In some embodiments a subject isa non-human animal that serves as a model for a disease or disorder thataffects humans. An animal model may be used, e.g., in preclinicalstudies, e.g., to assess efficacy and/or determine a suitable dose.

The term “small molecule” is used herein to refer to molecules, whethernaturally occurring or artificially created (e.g., via chemicalsynthesis) that have a relatively low molecular weight. Typically, asmall molecule is an organic compound. A small molecule may containmultiple carbon-carbon bonds, stereocenters, and other functional groups(e.g., amines, hydroxyl, carbonyls, heterocyclic rings, etc.). In someembodiments, a small molecule is monomeric. In some embodiments, a smallmolecule has a molecular weight of less than about 1500 g/mol. In someembodiments, a small molecule has a molecular weight of less than about1000 g/mol or less than about 500 g/mol. In certain embodiments a smallmolecule is a compound that has been deemed safe and effective for useas a diagnostic or therapeutic agent in humans or animals by anappropriate governmental agency or regulatory body.

As used herein, a “support” may be any entity or plurality of entitieshaving a surface to which a substance may be attached or on which asubstance may be placed. Examples, include, e.g., particles, slides,filters, interior wall or bottom of a vessel (e.g., a culture vesselsuch as a plate or flask, well of a microwell plate, tube), chips, etc.A support may be composed, e.g., of glass, metal, gels (e.g., agarose),ceramics, polymers, or combinations thereof.

The term “tumor” as used herein encompasses abnormal growths comprisingaberrantly proliferating cells. Tumors are typically characterized byexcessive cell proliferation that is not appropriately regulated (e.g.,that does not respond normally to physiological influences and signalsthat would ordinarily constrain proliferation) and may exhibit one ormore of the following properties: dysplasia (e.g., lack of normal celldifferentiation, resulting in an increased number or proportion ofimmature cells); anaplasia (e.g., greater loss of differentiation, moreloss of structural organization, cellular pleomorphism, abnormalitiessuch as large, hyperchromatic nuclei, high nuclear:cytoplasmic ratio,atypical mitoses, etc.); invasion of adjacent tissues (e.g., breaching abasement membrane); and/or metastasis. In certain embodiments a tumor isa malignant tumor, also referred to herein as a “cancer”. Malignanttumors have a tendency for sustained growth and an ability to spread,e.g., to invade locally and/or metastasize regionally and/or to distantlocations, whereas benign tumors often remain localized at the site oforigin and are often self-limiting in terms of growth. The term “tumor”includes malignant solid tumors (e.g., carcinomas, sarcomas) andmalignant growths in which there may be no detectable solid tumor mass(e.g., certain hematologic malignancies). The term “cancer” is generallyused interchangeably with “tumor” herein and/or to refer to a diseasecharacterized by one or more tumors, e.g., one or more malignant orpotentially malignant tumors. Cancer includes, but is not limited to:breast cancer; biliary tract cancer; bladder cancer; brain cancer (e.g.,glioblastomas, medulloblastomas); cervical cancer; choriocarcinoma;colon cancer; endometrial cancer; esophageal cancer; gastric cancer;hematological neoplasms including acute lymphocytic leukemia and acutemyelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma;hairy cell leukemia; chronic lymphocytic leukemia, chronic myelogenousleukemia, multiple myeloma; adult T-cell leukemia/lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastoma; melanoma, oral cancer includingsquamous cell carcinoma; ovarian cancer including ovarian cancer arisingfrom epithelial cells, stromal cells, germ cells and mesenchymal cells;neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer;sarcomas including angiosarcoma, gastrointestinal stromal tumors,leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; renal cancer including renal cell carcinoma and Wilmstumor; skin cancer including basal cell carcinoma and squamous cellcancer; testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullary carcinoma. It will be appreciated that a variety of differenttumor types can arise in certain organs, which may differ with regardto, e.g., clinical and/or pathological features and/or molecularmarkers. Tumors arising in a variety of different organs are discussed,e.g., in DeVita, Hellman, and Rosenberg's Cancer: Principles andPractice of Oncology (Cancer: Principles & Practice), LippincottWilliams & Wilkins; Ninth, North American Edition edition (May 16, 2011)or in the WHO Classification of Tumours series, 4^(th) ed, or 3^(rd) ed(Pathology and Genetics of Tumours series), by the International Agencyfor Research on Cancer (IARC), WHO Press, Geneva, Switzerland.

“Treat”, “treating” and similar terms refer to providing medical and/orsurgical management of a subject. Treatment may include, but is notlimited to, administering an agent or composition (e.g., apharmaceutical composition) to a subject. Treatment is typicallyundertaken in an effort to alter the course of a disease (which term isused to indicate any disease, disorder, or undesirable conditionwarranting therapy) in a manner beneficial to the subject. The effect oftreatment may include reversing, alleviating, reducing severity of,delaying the onset of, curing, inhibiting the progression of, and/orreducing the likelihood of occurrence or recurrence of the disease orone or more symptoms or manifestations of the disease. A therapeuticagent may be administered to a subject who has a disease or is atincreased risk of developing a disease relative to a member of thegeneral population. In some embodiments a therapeutic agent may beadministered to a subject who has had a disease but no longer showsevidence of the disease. The agent may be administered e.g., to reducethe likelihood of recurrence of evident disease. A therapeutic agent maybe administered prophylactically, i.e., before development of anysymptom or manifestation of a disease. “Prophylactic treatment” refersto providing medical and/or surgical management to a subject who has notdeveloped a disease or does not show evidence of a disease in order,e.g., to reduce the likelihood that the disease will occur or to reducethe severity of the disease should it occur. The subject may have beenidentified as being at risk of developing the disease (e.g., atincreased risk relative to the general population or as having a riskfactor that increases the likelihood of developing the disease.

A “variant” of a particular polypeptide or polynucleotide has one ormore alterations (e.g., additions, substitutions, and/or deletions) withrespect to a reference polypeptide or polynucleotide, which may bereferred to as the “original polypeptide” or “original polynucleotide”,respectively. An addition may be an insertion or may be at eitherterminus. A variant may be shorter or longer than the referencepolypeptide or polynucleotide. The term “variant” encompasses“fragments”. A “fragment” is a continuous portion of a polypeptide orpolynucleotide that is shorter than the reference polypeptide orpolynucleotide. In some embodiments a variant comprises or consists of afragment. In some embodiments a fragment or variant is at least 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, ormore as long as the reference polypeptide or polynucleotide. In someembodiments a fragment may lack an N-terminal and/or C-terminal portionof a reference polypeptide. For example, a fragment may lack up to 5%,10%, 15%, 20%, or 25% of the length of the polypeptide from either orboth ends. A fragment may be an N-terminal, C-terminal, or internalfragment. In some embodiments a variant polypeptide comprises orconsists of at least one domain of a reference polypeptide. In someembodiments a variant polynucleotide hybridizes to a referencepolynucleotide under art-recognized stringent conditions, e.g., highstringency conditions, for sequences of the length of the referencepolypeptide. In some embodiments a variant polypeptide or polynucleotidecomprises or consists of a polypeptide or polynucleotide that is atleast 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or moreidentical in sequence to the reference polypeptide or polynucleotideover at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% of the reference polypeptide or polynucleotide. Insome embodiments a variant polypeptide comprises or consists of apolypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or more identical in sequence to the reference polypeptideover at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% of the reference polypeptide, with the proviso that,for purposes of computing percent identity, a conservative amino acidsubstitution is considered identical to the amino acid it replaces. Insome embodiments a variant polypeptide comprises or consists of apolypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or more identical to the reference polypeptide over at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%of the reference polypeptide, with the proviso that any one or moreamino acid substitutions (up to the total number of such substitutions)may be restricted to conservative substitutions. In some embodiments apercent identity is measured over at least 100; 200; 300; 400; 500; 600;700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000;4,500; or 5,000 amino acids. In some embodiments the sequence of avariant polypeptide comprises or consists of a sequence that has N aminoacid differences with respect to a reference sequence, wherein N is anyinteger between 1 and 10 or between 1 and 20 or any integer up to 1%,2%, 5%, or 10% of the number of amino acids in the referencepolypeptide, where an “amino acid difference” refers to a substitution,insertion, or deletion of an amino acid. In some embodiments adifference is a conservative substitution. Conservative substitutionsmay be made, e.g., on the basis of similarity in side chain size,polarity, charge, solubility, hydrophobicity, hydrophilicity and/or theamphipathic nature of the residues involved. In some embodiments,conservative substitutions may be made according to Table A, whereinamino acids in the same block in the second column and in the same linein the third column may be substituted for one another other in aconservative substitution. Certain conservative substitutions aresubstituting an amino acid in one row of the third column correspondingto a block in the second column with an amino acid from another row ofthe third column within the same block in the second column.

TABLE A Aliphatic Non-polar GAP ILV Polar—uncharged CSTM NQPolar—charged DE KR Aromatic HPWY

In some embodiments, proline (P) is considered to be in an individualgroup. In some embodiments, cysteine (C) is considered to be in anindividual group. In some embodiments, proline (P) and cysteine (C) areeach considered to be in an individual group. Within a particular group,certain substitutions may be of particular interest in certainembodiments, e.g., replacements of leucine by isoleucine (or viceversa), serine by threonine (or vice versa), or alanine by glycine (orvice versa).

In some embodiments a variant is a functional variant, i.e., the variantat least in part retains at least one activity of the referencepolypeptide or polynucleotide. In some embodiments a variant at least inpart retains more than one or substantially all known activities of thereference polypeptide or polynucleotide. An activity may be, e.g., acatalytic activity, binding activity, ability to perform or participatein a biological function or process, etc. In some embodiments anactivity is one that has (or the lack of which has) a detectable effecton an observable phenotype of a cell or organism. In some embodiments anactivity of a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or more, of the activity of the reference polypeptide orpolynucleotide, up to approximately 100%, approximately 125%, orapproximately 150% of the activity of the reference polypeptide orpolynucleotide, in various embodiments. In some embodiments a variant,e.g., a functional variant, comprises or consists of a polypeptide atleast 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%. 99.5% or 100% identicalto an reference polypeptide or polynucleotide over at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the full length ofthe reference polypeptide or polynucleotide or over at least 70%, 75%,80%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99% or 100% of a functionalfragment of the reference polypeptide or polynucleotide. In someembodiments an alteration, e.g., a substitution or deletion, e.g., in afunctional variant, does not alter or delete an amino acid or nucleotidethat is known or predicted to be important for an activity, e.g., aknown or predicted catalytic residue or residue involved in binding asubstrate or cofactor. In some embodiments nucleotide(s), amino acid(s),or region(s) exhibiting lower degrees of conservation across species ascompared with other amino acids or regions may be selected foralteration. Variants may be tested in one or more suitable assays toassess activity. In certain embodiments a polypeptide or polynucleotidesequence in the NCBI RefSeq database may be used as a referencesequence. In some embodiments a variant or fragment of a naturallyoccurring polypeptide or polynucleotide is a naturally occurring variantor fragment. In some embodiments a variant or fragment of a naturallyoccurring polypeptide or polynucleotide is not naturally occurring.Calculations of sequence identity can be performed as follows. Sequencesare aligned for optimal comparison purposes and gaps can be introducedin one or both of a first and a second sequence for optimal alignment.When a position in the first sequence is occupied by the same residue asthe corresponding position in the second sequence, the sequences aredeemed to be identical at that position. The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps, and thelength of each gap, introduced for optimal alignment of the twosequences. Sequences can be aligned and/or percent identity determinedwith the use of a variety of algorithms and computer programs known inthe art. For example, computer programs such as BLAST2, BLASTN, BLASTP,Gapped BLAST, etc., may be used to generate alignments and/or to obtaina percent identity. The algorithm of Karlin and Altschul (Karlin andAltschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990) modified as inKarlin and Altschul, Proc. Natl. Acad Sci. USA 90:5873-5877, 1993 isincorporated into the NBLAST and XBLAST programs of Altschul et al.(Altschul, et al., J. MoI. Biol. 215:403-410, 1990). In someembodiments, to obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (Altschul, et al.Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs may beused. See the Web site having URL www.ncbi.nlm.nih.gov and/or McGinnis,S. and Madden, T L, W20-W25 Nucleic Acids Research, 2004, Vol. 32, Webserver issue. Other suitable programs include CLUSTALW (Thompson J D,Higgins D G, Gibson T J, Nuc Ac Res, 22:4673-4680, 1994) and GAP (GCGVersion 9.1; which implements the Needleman & Wunsch, 1970 algorithm(Needleman S B, Wunsch C D, J Mol Biol, 48:443-453, 1970.) The percentidentity between a sequence of interest A and a second sequence B may becomputed by aligning the sequences, allowing the introduction of gaps tomaximize identity, determining the number of residues (nucleotides oramino acids) that are opposite an identical residue, dividing by theminimum of TGA and TGB (here TGA and TGB are the sum of the number ofresidues and internal gap positions in sequences A and B in thealignment), and multiplying by 100. Percent identity may be evaluatedover a window of evaluation. In some embodiments a window of evaluationmay have a length of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, ormore, e.g., 100%, of the length of the shortest of the sequences beingcompared. In some embodiments a window of evaluation is at least 100;200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000;2,500; 3,000; 3,500; 4,000; 4,500; or 5,000 amino acids. In someembodiments no more than 20%, 10%, 5%, or 1% of positions in eithersequence or in both sequences over a window of evaluation are occupiedby a gap. In some embodiments no more than 20%, 10%, 5%, or 1% ofpositions in either sequence or in both sequences are occupied by a gap.

A “vector” may be any of a number of nucleic acid molecules or virusesor portions thereof that are capable of mediating entry of, e.g.,transferring, transporting, etc., a nucleic acid of interest betweendifferent genetic environments or into a cell. The nucleic acid ofinterest may be linked to, e.g., inserted into, the vector using, e.g.,restriction and ligation. Vectors include, for example, DNA or RNAplasmids, cosmids, naturally occurring or modified viral genomes orportions thereof, nucleic acids that can be packaged into viral capsids,mini-chromosomes, artificial chromosomes, transposons (e.g., SleepingBeauty transposon), etc. Plasmid vectors typically include an origin ofreplication (e.g., for replication in prokaryotic cells). A plasmid mayinclude part or all of a viral genome (e.g., a viral promoter, enhancer,processing or packaging signals, and/or sequences sufficient to giverise to a nucleic acid that can be integrated into the host cell genomeand/or to give rise to infectious virus). Viruses or portions thereofthat can be used to introduce nucleic acids into cells may be referredto as viral vectors. Viral vectors include, e.g., adenoviruses,adeno-associated viruses, retroviruses (e.g., lentiviruses, gammaretroviruses), vaccinia virus and other poxviruses, herpesviruses (e.g.,herpes simplex virus), and others. Viral vectors may or may not containsufficient viral genetic information for production of infectious viruswhen introduced into host cells, i.e., viral vectors may bereplication-competent or replication-defective. In some embodiments,e.g., where sufficient information for production of infectious virus islacking, it may be supplied by a host cell or by another vectorintroduced into the cell, e.g., if production of virus is desired. Insome embodiments such information is not supplied, e.g., if productionof virus is not desired. A nucleic acid to be transferred may beincorporated into a naturally occurring or modified viral genome or aportion thereof or may be present within a viral capsid as a separatenucleic acid molecule. A vector may contain one or more nucleic acidsencoding a marker suitable for identifying and/or selecting cells thathave taken up the vector. Markers include, for example, various proteinsthat increase or decrease either resistance or sensitivity toantibiotics or other agents (e.g., a protein that confers resistance toan antibiotic such as puromycin, hygromycin or blasticidin), enzymeswhose activities are detectable by assays known in the art (e.g.,β-galactosidase or alkaline phosphatase), and proteins or RNAs thatdetectably affect the phenotype of cells that express them (e.g.,fluorescent proteins). Vectors often include one or more appropriatelypositioned sites for restriction enzymes, which may be used tofacilitate insertion into the vector of a nucleic acid, e.g., a nucleicacid to be expressed. An expression vector is a vector into which adesired nucleic acid has been inserted or may be inserted such that itis operably linked to regulatory elements (also termed “regulatorysequences”, “expression control elements”, or “expression controlsequences”) and may be expressed as an RNA transcript (e.g., an mRNAthat can be translated into protein or a noncoding RNA such as an shRNAor miRNA precursor). Expression vectors include regulatory sequence(s),e.g., expression control sequences, sufficient to direct transcriptionof an operably linked nucleic acid under at least some conditions; otherelements required or helpful for expression may be supplied by, e.g.,the host cell or by an in vitro expression system. Such regulatorysequences typically include a promoter and may include enhancersequences or upstream activator sequences. In some embodiments a vectormay include sequences that encode a 5′ untranslated region and/or a 3′untranslated region, which may comprise a cleavage and/orpolyadenylation signal. In general, regulatory elements may be containedin a vector prior to insertion of a nucleic acid whose expression isdesired or may be contained in an inserted nucleic acid or may beinserted into a vector following insertion of a nucleic acid whoseexpression is desired. As used herein, a nucleic acid and regulatoryelement(s) are said to be “operably linked” when they are covalentlylinked so as to place the expression or transcription of the nucleicacid under the influence or control of the regulatory element(s). Forexample, a promoter region would be operably linked to a nucleic acid ifthe promoter region were capable of effecting transcription of thatnucleic acid. One of ordinary skill in the art will be aware that theprecise nature of the regulatory sequences useful for gene expressionmay vary between species or cell types, but may in general include, asappropriate, sequences involved with the initiation of transcription,RNA processing, or initiation of translation. The choice and design ofan appropriate vector and regulatory element(s) is within the abilityand discretion of one of ordinary skill in the art. For example, one ofskill in the art will select an appropriate promoter (or otherexpression control sequences) for expression in a desired species (e.g.,a mammalian species) or cell type. A vector may contain a promotercapable of directing expression in mammalian cells, such as a suitableviral promoter, e.g., from a cytomegalovirus (CMV), retrovirus, simianvirus (e.g., SV40), papilloma virus, herpes virus or other virus thatinfects mammalian cells, or a mammalian promoter from, e.g., a gene suchas EF1alpha, ubiquitin (e.g., ubiquitin B or C), globin, actin,phosphoglycerate kinase (PGK), etc., or a composite promoter such as aCAG promoter (combination of the CMV early enhancer element and chickenbeta-actin promoter). In some embodiments a human promoter may be used.In some embodiments, a promoter that ordinarily directs transcription bya eukaryotic RNA polymerase I (a “pol I promoter”), e.g., a promoter fortranscription of ribosomal RNA (other than 5S rRNA) may be used. In someembodiments, a promoter that ordinarily directs transcription by aeukaryotic RNA polymerase II (a “pol II promoter”) or a functionalvariant thereof is used. In some embodiments, a promoter that ordinarilydirects transcription by a eukaryotic RNA polymerase III (a “pol IIIpromoter”), e.g., a promoter for transcription of U6, H1, 7SK or tRNApromoter or a functional variant thereof) or a functional variantthereof is used. One of ordinary skill in the art will select anappropriate promoter for directing transcription of a sequence ofinterest. Examples of expression vectors that may be used in mammaliancells include, e.g., the pcDNA vector series, pSV2 vector series, pCMVvector series, pRSV vector series, pEF1 vector series, Gateway® vectors,etc. Examples of virus vectors that may be used in mammalian cellsinclude, e.g., adenoviruses, adeno-associated viruses, poxviruses suchas vaccinia viruses and attenuated poxviruses, retroviruses (e.g.,lentiviruses), Semliki Forest virus, Sindbis virus, etc. In someembodiments, regulatable (e.g., inducible or repressible) expressioncontrol element(s), e.g., a regulatable promoter, is/are used so thatexpression can be regulated, e.g., turned on or increased or turned offor decreased. For example, the tetracycline-regulatable gene expressionsystem (Gossen & Bujard, Proc. Natl. Acad. Sci. 89:5547-5551,1992) orvariants thereof (see, e.g., Allen, N, et al. (2000) Mouse Genetics andTransgenics: 259-263; Urlinger, S, et al. (2000). Proc. Natl. Acad. Sci.U.S.A. 97 (14): 7963-8; Zhou, X., et al (2006). Gene Ther. 13 (19):1382-1390 for examples) can be employed to provide inducible orrepressible expression. Other inducible/repressible systems may be usedin various embodiments. For example, expression control elements thatcan be regulated by small molecules such as artificial or naturallyoccurring hormone receptor ligands (e.g., steroid receptor ligands suchas naturally occurring or synthetic estrogen receptor or glucocorticoidreceptor ligands), tetracycline or analogs thereof, metal-regulatedsystems (e.g., metallothionein promoter) may be used in certainembodiments. In some embodiments, tissue-specific or cell type specificregulatory element(s) may be used, e.g., in order to direct expressionin one or more selected tissues or cell types.

In some embodiments a vector is used to insert exogenous DNA into thegenome of a cell. In general, any suitable vector may be used. In someembodiments the vector is a viral vector, e.g., a retroviral vector suchas a lentiviral vector or gamma retroviral vector, or an adenoviral orAAV vector. In some embodiments the vector is a plasmid, e.g., a DNAplasmid. In some embodiments, the plasmid comprises DNA to be insertedinto the genome of a cell, wherein the DNA is located between bindingsites for a transposase (“transposase binding sites”) so thatintegration of the DNA can be achieved by supplying the transposase,e.g., by expressing it from the same or a different plasmid. In someembodiments the transposase is e.g., a member of the Sleeping Beautyfamily of transposases, the piggyBac family of transposases, or the Tol2family of transposases (see Grabundzija, I., et al., Molecular Therapy,vol. 18 no. 6, 1200-1209 (2010) for review of transposon systems thatutilize these transposases, and various uses thereof in geneticengineering). Examples of Sleeping Beauty transposases include SB10,SB11, and SB100X (see, e.g., Mates, L., et al., Nat Genet. (2009)41(6):753-61; Jin, Z., et al. Gene Therapy (2011) 18, 849-856). In someembodiments the vector is suitable for use to genetically engineercells, e.g., human cells, that are to be administered to a humansubject. In some embodiments the vector has been used in at least oneclinical trial in human subjects, results of which have been published,without reported clinically unacceptable adverse events attributable tothe vector. In some embodiments the vector is a self-inactivatingretroviral vector. Such vectors may be created by deletion of at leastpart of the U3 portion of the 3′ LTR. Exemplary retroviral andlentiviral vectors are described in US Pat. Pub. No. 20050251872, USPat. Pub. No. 20040259208, and various other references cited herein. Insome embodiments a second or third generation lentiviral vector may beused.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS I. SortaggingNon-Genetically Engineered Eukaryotic Polypeptides and Cells

The present disclosure describes the unexpected discovery thatnon-genetically engineered mammalian cells can be effectively labeledusing sortase, i.e., in a sortase-catalyzed transacylation. In someaspects, the invention provides methods of using sortase to conjugateagents to living mammalian cells that have not been geneticallyengineered to express a protein comprising a sortase recognition motifor a nucleophilic acceptor sequence. In some embodiments the mammaliancells have not been genetically engineered. Some aspects of thisinvention relate to the recognition that the sortase-catalyzedtransacylation reaction allows for the conjugation of agents to one ormore polypeptides that are endogenous to living mammalian cells, i.e.,sortase can be used to conjugate agents to living mammalian cells thatare not genetically engineered for sortagging. As used herein, apolypeptide is “not genetically engineered for sortagging” if thepolypeptide is not genetically engineered in a way that allows it toserve as a sortase substrate or as a nucleophile in a sortase-catalyzedreaction, i.e., the polypeptide is not genetically engineered tocomprise a sortase recognition motif in a region accessible to a sortase(e.g., at or near the C-terminus) and is not genetically engineered tocomprise a nucleophilic acceptor sequence that can serve as anucleophile in a sortase-catalyed reaction, such as a sequencecomprising one or more glycines, located at the N-terminus of thepolypeptide or positioned such that cleavage of the polypeptide canresult in the sequence being located at an N-terminus. In someembodiments the polypeptide is not genetically engineered. A cell isconsidered “not genetically engineered for sortagging” if the cell hasnot been genetically engineered to express a polypeptide that (eithernaturally or as a result of genetic engineering) is suitable to serve asa sortase substrate or as a nucleophile in a sortase-catalyzed reaction.In some embodiments the cell has not been genetically engineered toexpress a polypeptide comprising a sortase recognition motif or anucleophilic acceptor sequence. In some embodiments the cell is notgenetically engineered. In some embodiments the cell does not comprise amodification to its genome introduced by the hand of man. In someaspects, the invention relates to use of sortase to attach any of a widevariety of agents to the surface of non-genetically engineered mammaliancells. Unless otherwise indicated or clearly evident from the context,where the present disclosure refers to sortagging mammalian cells it isgenerally intended to mean mammalian cells that have not beengenetically engineered for sortagging. In certain embodiments the animalcells are not genetically engineered.

As described in Examples 1 and 2, non-genetically engineered mousesplenocytes were effectively sortagged with a variety of sortasesubstrates at readily detectable levels. In other experiments,sortagging of non-genetically engineered cells of a human kidney cellline (HEK293T cells) and canine kidney cell line (MDCK cells) andvariety of other non-genetically engineered eukaryotic cell types(fungal, protozoal) was also observed. Thus, sortase can be used formodification of mammalian cell surfaces and other eukaryotic cellsurfaces without requiring that the cells be engineered to expresspolypeptides comprising a sortase recognition sequence or nucleophilicacceptor sequence. In accordance with certain embodiments of the presentinvention a non-genetically engineered mammalian cell expresses one ormore endogenous polypeptides comprising a nucleophilic acceptorsequence, thus allowing it to serve as a nucleophile in asortase-catalyzed transacylation. Such polypeptide(s) may comprise asequence of one or more glycines exposed at the cell surface, e.g., inan N-terminal domain, available to act as a nucleophile in a reaction inwhich sortase is used to conjugate a sortase substrate to thepolypeptide. In some embodiments the endogenous polypeptide may comprisean N-terminal glycine. In some embodiments the endogenous polypeptidemay comprise a sequence of between 1-10 glycines at its N-terminus,e.g., 1, 2, 3, 4, or 5 glycines. In some embodiments the polypeptide maynot have an N-terminal glycine when initially synthesized (e.g., theN-terminal amino acid may be methionine) but undergoes co-translationalor post-translational processing (e.g., cleavage) or partial degradationso that a sequence of one or more glycines is present at the N-terminus.For example, a secretion signal sequence may be removed. Such processingor degradation may occur before exposure of at least a portion of thepolypeptide at the cell surface (e.g., in the endoplasmic reticulum) ormay occur following exposure of at least a portion of the polypeptide tothe extracellular environment. It will thus be understood that aspectsof the invention comprise sortagging eukaryotic cell surfaces, e.g.,mammalian cell surfaces, without first modifying the cells so as tocause them to have a sortase recognition sequence or a moiety capable ofserving as a nucleophile in a sortase-catalyzed reaction attached totheir surface. Aspects of the invention comprise sortagging anendogenous polypeptide expressed by a living eukaryotic cell, e.g., amammalian cell, wherein the endogenous polypeptide comprises anextracellular domain that naturally comprises one or more amino acidscapable of serving as a nucleophile in a sortase-catalyzed reaction.According to such aspects the natural DNA sequence encoding theextracellular domain of such polypeptide has not been modified by thehand of man to encode an amino acid capable of serving as a nucleophilein a sortase-catalyzed reaction, and the polypeptide has not beenmodified by the hand of man by adding such an amino acid to theextracellular domain. For example, the extracellular domain of thepolypeptide has not been subjected to covalent or noncovalent linkage ofa (G)n moiety, an (A)n moiety, or a moiety comprising a free aminecapable of serving as a nucleophile in a sortase-catalyzed reaction. Incertain embodiments of any aspect, cells are not subjected to chemicalmodification prior to sortagging.

In some aspects, the invention provides compositions useful forgenerating sortase-modified eukaryotic cells, e.g., sortase-modifiedmammalian cells. In some embodiments the compositions comprise a sortaseand one or more living eukaryotic cells, e.g., mammalian cells, whereinthe cell(s) do not express a polypeptide that has been geneticallyengineered to comprise a sortase recognition motif or nucleophilicacceptor sequence. In some embodiments the cell(s) are not geneticallyengineered. In some embodiments a composition further comprises asortase substrate. In some embodiments the sortase substrate comprisesany of a variety of agents, e.g.,

In some aspects, the invention provides compositions comprisingsortase-modified eukaryotic cells, e.g., sortase-modified mammaliancells. In some embodiments the compositions comprise one or moresortase-modified eukaryotic cells, e.g., sortase-modified mammaliancells, wherein the cells are modified by conjugation of an agent to apolypeptide expressed by the cells, wherein the polypeptide has not beengenetically engineered to comprise a sortase recognition motif ornucleophilic acceptor sequence. In some embodiments the eukaryoticcells, e.g., mammalian cells, are not genetically engineered.

In some aspects, the invention provides a eukaryotic cell, e.g., amammalian cell, that comprises an agent conjugated via a sortaserecognition motif to a non-genetically engineered endogenous polypeptideexpressed by the cell. In some embodiments the cell is not geneticallyengineered. In some embodiments, two, three, four or more differentnon-genetically engineered endogenous polypeptides expressed by the cellhave an agent conjugated thereto via a sortase recognition motif. Theagents attached to different polypeptides may be the same or the cellmay be sortagged with multiple different agents.

In some aspects, the invention provides methods of generatingsortase-modified eukaryotic cells, e.g., mammalian cells. In someaspects, the invention provides methods that comprise conjugating anagent to a non-genetically engineered eukaryotic, e.g., mammalian,polypeptide using a sortase. In some aspects, the invention providesmethods comprising conjugating an agent to a mammalian polypeptide usinga sortase, wherein the polypeptide has not been engineered to comprise asortase recognition motif or nucleophilic acceptor sequence. In someembodiments the polypeptide is expressed by a living mammalian cell, andthe methods comprise contacting the cell with a sortase and a sortasesubstrate comprising the agent under conditions suitable for a sortasereaction to occur. In some embodiments the polypeptide comprises anextracellular domain, and the methods comprise conjugating the sortasesubstrate to the extracellular domain of the polypeptide. In someembodiments the extracellular domain comprises the N-terminus of thepolypeptide. In some embodiments the mammalian polypeptide comprises anN-terminal nucleophilic acceptor sequence, e.g., a sequence comprisingan N-terminal glycine, before conjugation of the sortase substratethereto. In some embodiments a method comprises contacting one or moreliving mammalian cells with sortase and a sortase substrate underconditions and for a time suitable for a sortase-mediated transacylationreaction to occur, wherein the living mammalian cell(s) have not beengenetically engineered to express a polypeptide that comprises a sortaserecognition motif or nucleophilic acceptor sequence. In some embodimentsthe mammalian cell(s) have not been genetically engineered.

In some embodiments a method further comprises separating one or more ofthe living mammalian cell(s) from sortase and/or from sortase substratethat is not conjugated to the cells. The cells may be processed so as toachieve a selected degree of purity with respect to sortase,unconjugated sortase substrate, or both. For example, in someembodiments the amount of sortase and/or the amount of unconjugatedsortase substrate may be reduced to below a selected concentrationand/or a selected proportion of the sortase and/or unconjugated sortasesubstrate may be removed. For example, the concentration of sortaseand/or unconjugated sortase substrate may be reduced by at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more 99.9% relative to theinitial concentration, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, or more of the sortase and/or unconjugated sortasesubstrate that was present in the composition comprising one or moreliving mammalian cells and sortase may be removed. In some embodimentsthe concentration of sortase and/or unconjugated sortase substrate isreduced to no more than 0.01%, 0.05%, 0.1%, 0.5%, or 1.0% relative tothe concentration used to sortag the mammalian cells. In someembodiments a sortase polypeptide and/or unconjugated sortase substrateis not detectable in the composition as measured by standard immunoblotusing an antibody or other affinity agent that specifically binds to thepolypeptide and/or agent. Various suitable methods for separatingsortagged cells from sortase and/or from unconjugated sortase substrateare described herein, but other suitable methods may be used.

In some aspects, the invention provides living mammalian cells having anagent conjugated thereto via a sortase-mediated transacylation reaction(“sortagged cells”). In some embodiments the agent is conjugated to apolypeptide comprising a domain exposed at the cell surface. In someembodiments compositions comprising a plurality of such cells areprovided. In some embodiments the polypeptide to which the agent isconjugated comprises, after such conjugation, a sortase recognitionmotif. In some embodiments a composition comprising a plurality of suchsortagged cells has a reduced level of sortase, unconjugated sortasesubstrate, or both, as compared with a composition in which the cellswere sortagged. In some embodiments the composition has a selecteddegree of purity with respect to sortase, unconjugated sortasesubstrate, or both. In some embodiments at least a selected percentageof the cells in a composition are modified, i.e., have an agentconjugated thereto by sortase. For example, in some embodiments at least5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or more of the cells have an agent conjugated thereto. Insome embodiments a method may comprise separating cells that have anagent conjugated thereto from cells that do not.

In certain embodiments an endogenous mammalian polypeptide comprises oneor more N-terminal glycines. A polypeptide comprising one or moreN-terminal glycines may be represented as G(G)n-B¹, wherein G isglycine, B¹ represents an amino acid sequence, and n is a non-negativeinteger, e.g., between 0 and 10, or may equivalently be represented asfollows:

wherein B¹ represents an amino acid sequence, and n is a non-negativeinteger, e.g., between 0 and 10. In certain embodiments n is 0, 1, 2, 3,4, or 5. In general, B¹ may be of any length and sequence, providedthat, in certain embodiments, the polypeptide comprising B¹ has asequence that is endogenous to a mammalian cell, so that a mammaliancell may express the polypeptide without having been geneticallyengineered to do so.

In some embodiments the invention provides a method comprisingcontacting a living mammalian cell that comprises a polypeptide of thefollowing structure exposed at the cell surface:

with a sortase substrate of the following structure:

wherein the transamidase recognition sequence is an amino acid sequencemotif recognized by a transamidase enzyme;

X is —O—, —NR—, or —S—; wherein R is hydrogen, substituted orunsubstituted aliphatic, or substituted or unsubstitutedheteroaliphatic;

A¹ is acyl, substituted or unsubstituted aliphatic, substituted orunsubstituted heteroaliphatic, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, an amino acid, a peptide, aprotein, a polynucleotide, a carbohydrate, a tag, a metal atom, acontrast agent, a catalyst, a non-polypeptide polymer, a recognitionelement, a small molecule, a lipid, a linker, a label, an epitope, anantigen, a therapeutic agent, a toxin, a radioisotope, a particle;

R¹ is acyl, substituted or unsubstituted aliphatic, substituted orunsubstituted heteroaliphatic, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; in the presence of atransamidase enzyme, for example, a sortase, under suitable conditionsto form a compound of formula:

wherein n is between 0 and 10, and wherein B¹ represents anextracellular domain of a polypeptide expressed by a living mammaliancell.

The resulting sortase-modified cell may be represented as follows:

wherein the circle represents a cell and the short line between B¹ andthe cell indicates that B¹ is attached to the cell (e.g., B¹ may be partof an integral membrane polypeptide or peripheral membrane polypeptide).The sortase substrate and agent A¹ may be said to be conjugated to thecell. It will be appreciated that the XR¹ moiety of the sortasesubstrate is released as a reaction byproduct.

In some embodiments X is —NR— and XR¹ represents glycine (G), alanine(A), or another amino acid that may be found at the C-terminus of anaturally occurring sortase recognition sequence. In some embodiments Xis —NR— and XR¹ represents (G)_(j)(Xaa)_(m), wherein each Xaa can beindependently any amino acid, j is at least 1, and j+m is between 1 and5, between 1 and 10, between 1 and 20, or between 1, 5, 10, or 20 and100. In some embodiments j is 1. In some embodiments j is 2, 3, 4, or 5.In some embodiments j is 1. In some embodiments j is 2, 3, 4, or 5. Insome embodiments XR¹ comprises a detectable label or epitope tag (e.g.,(Xaa)_(m) may comprise an epitope tag or may have a tag or labelattached to a side chain), so that the reaction byproduct may bedetected and/or isolated or separated from the cells.

In certain embodiments X is —O—, —NR—, or —S—; wherein R is hydrogen,substituted or unsubstituted aliphatic, or substituted or unsubstitutedheteroaliphatic. In certain embodiments R¹ is acyl. In certainembodiments R¹ is substituted aliphatic. In certain embodiments, R¹ isunsubstituted aliphatic. In some embodiments, R¹ is substituted C₁₋₁₂aliphatic. In some embodiments, R¹ is unsubstituted C₁₋₁₂ aliphatic. Insome embodiments, R¹ is substituted C₁₋₆ aliphatic. In some embodiments,R¹ is unsubstituted C₁₋₆ aliphatic. In some embodiments, R¹ is C₁₋₃aliphatic. In some embodiments, R¹ is butyl. In some embodiments, R¹ isn-butyl. In some embodiments, R¹ is isobutyl. In some embodiments, R¹ ispropyl. In some embodiments, R¹ is n-propyl. In some embodiments, R¹ isisopropyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ ismethyl. In certain embodiments, R¹ is substituted aryl. In certainembodiments, R¹ is unsubstituted aryl. In certain embodiments, R¹ issubstituted phenyl. In certain embodiments, R¹ is unsubstituted phenyl.In certain embodiments R¹ comprises a label (e.g., a fluorophore) oraffinity tag. In certain embodiments a label or affinity tag may beused, e.g., to detect and/or remove sortase substrate that does notparticipate in a sortase-mediated reaction, to detect and/or removereaction byproduct comprising XR¹, to measure or monitor the progress ofa sortase-mediated reaction or determine the extent to which sortasesubstrate has been consumed.

In certain embodiments, the C-terminal amino acid of a 5 amino acidtransamidase recognition sequence, e.g., a transamidase recognitionsequence that would ordinarily comprise a C-terminal glycine or alanineas a fifth amino acid, may be omitted. For example, an acyl group

that is not a glycine, alanine, or other residue that may be found atthe C-terminus of a naturally occurring transamidase recognitionsequence may replace the C-terminal amino acid of a 5 amino acidtransamidase recognition sequence. In some embodiments, XR¹ is selectedto be a moiety that exhibits poor nucleophilicity once released from thetransamidase, thereby providing for a more efficient ligation, e.g., ascompared with the efficiency if XR¹ is a C-terminal amino acid of anaturally occurring transamidase recognition sequence, e.g., glycine.Any moiety exhibiting such poor nucleophilicity can be used inaccordance with certain embodiments. In some embodiments, the acyl group

is not an amino acid or peptide. In some embodiments, the acyl group is

In some embodiments, the acyl group is

Some embodiments of the invention provide modified, non-geneticallyengineered mammalian proteins comprising a sortase recognition motif.Some embodiments provide modified, non-genetically engineered mammalianproteins comprising a sortase recognition motif having an agentconjugated thereto. Some embodiments provide mammalian cells comprisinga modified, non-genetically engineered protein comprising a sortaserecognition motif. Some embodiments provide mammalian cells comprising amodified, non-genetically engineered protein comprising a sortaserecognition motif having an agent conjugated thereto.

Some embodiments provide a non-genetically engineered mammalian proteincomprising an N-terminal modification installed by sortase, wherein thenon-genetically engineered mammalian protein comprises a structureaccording to Formula (I):

[Xaa]_(y)-TRS-PRT  (I)

Some embodiments provide a non-genetically engineered mammalian proteincomprising an N-terminal modification installed by sortase, wherein thenon-genetically engineered mammalian protein comprises a structureaccording to Formula (II):

M-[Xaa]_(y)-TRS-PRT  (II)

In Formulas (I) and (II):

PRT is an amino acid sequence of at least three amino acids, wherein thesequence is endogenous to a mammalian cell, e.g., a polypeptidecomprising B¹ as described above;

each instance of Xaa is independently any amino acid residue;

y is 0 or an integer between 1-2000

TRS is a transamidase recognition motif; and

M in Formula II is an agent attached to [Xaa]_(y) or, if y is 0, M is amoiety directly attached to the TRS. In some embodiments M comprises anamino acid, a peptide, a protein, a polynucleotide, a carbohydrate, atag, a metal atom, a contrast agent, a catalyst, a non-polypeptidepolymer, a recognition element, a small molecule, a lipid, a linker, alabel, an epitope, an antigen, a therapeutic agent, a toxin, aradioisotope, a click chemistry handle, or a particle.

In some aspects, a mammalian cell comprises a modified protein accordingto Formula (I) or Formula (II). In some embodiments at least the portionof the protein comprising [Xaa]_(y)-TRS or comprising M-[Xaa]_(y)-TRS isexposed at the cell surface. In some embodiments the cell is notgenetically engineered.

In some embodiments a polypeptide modified by sortase (e.g., apolypeptide comprising B¹ as described above) is an integral membraneprotein (IMP) or a subunit of an IMP. An IMP is a protein that isnaturally stably attached to the plasma membrane of a cell. Apolypeptide may be attached to the cell in any of various ways by whichmammalian polypeptides are naturally attached to cell plasma membranes.An IMP may comprise a transmembrane (TM) domain and, in some embodimentsan intracellular domain. A polypeptide may have its C-terminal aminoacid located within the plasma membrane or in the cytosol. A TMpolypeptide may be a single pass TM polypeptide or multi-pass. In someembodiments a polypeptide is associated with the membrane from one sidebut does not span the lipid bilayer completely, may bind covalently to amembrane lipid, may have a glycophosphatidylinositol (GPI) anchor,and/or may be associated with membrane lipids via electrostatic or ionicinteractions. In some embodiments a polypeptide modified by sortase is aperipheral membrane protein.

Sortase substrates may comprise any of a wide variety of agents, e.g.,an amino acid, a peptide, a protein, a polynucleotide, a carbohydrate, atag, a metal atom, a contrast agent, a catalyst, a non-polypeptidepolymer, a recognition element, a small molecule, a lipid, a linker, alabel, an epitope, an antigen, a therapeutic agent, a toxin, aradioisotope, a particle, or a click chemistry handle. In certainembodiments an agent may comprise two or more such moieties. Forexample, a linker may have any of a wide variety of moieties attachedthereto.

In some embodiments a sortase substrate to be used to conjugate an agentA¹ to a mammalian cell using sortase may be represented as follows:

wherein X and R¹ are as described above.

In some embodiments A¹ comprises a protein. In some embodiments, A¹comprises a peptide. In some embodiments, A¹ comprises an amino acidsequence comprising at least 3 amino acids. In some embodiments, A¹comprises an antibody, an antibody chain, an antibody fragment, anantigen-binding antibody domain, a VHH domain, a single-domain antibody,a camelid antibody, a nanobody, an adnectin, an affibody, an anticalin,or an aptamer. In some embodiments, A¹ comprises a recombinant protein,a protein or peptide comprising one or more non-standard amino acids(e.g., D-amino acids), a branched protein or peptide, a therapeuticprotein or peptide, an enzyme, a polypeptide subunit of a multisubunitprotein, a transmembrane protein, a cell surface protein, a methylatedpeptide or protein, an acylated peptide or protein, a lipidated peptideor protein, a phosphorylated peptide or protein, or a glycosylatedpeptide or protein. In some embodiments, A¹ comprises an antigen or anepitope. In some embodiments A¹ comprises an enzyme, growth factor,cytokine, costimulator, or adjuvant. In some embodiments A¹ comprises asmall molecule, a click chemistry handle, a fatty acid, apolynucleotide, a carbohydrate, a tag, a metal atom, a contrast agent, apeptide, a polypeptide, a non-polypeptide polymer, a recognitionelement, a lipid, a label, or a particle. In some embodiments A¹comprises a binding moiety. In some embodiments A¹ comprises a targetingmoiety. In some embodiments a moiety may be incorporated into A¹ in anymanner and at any position that can be envisioned by those of ordinaryskill in the art. For example, A¹ may comprise an amino acid, and amoiety may be attached, e.g., to the central carbon of the amino acid,the side chain of the amino acid, the carboxyl group of the amino acid,or the nitrogen. In some embodiments an agent comprises an amino acidhaving a side chain comprising a primary or secondary amine. Examples ofsuitable amino acids include, e.g., lysine, ε-aminocaproic acid, andvarious others known in the art. A sortase recognition motif may beextended to include such an amino acid, e.g., as K-LPXTG. Such aminoacids may conveniently be used as a point of attachment of a moiety ofinterest through reaction with the amine group. In some embodiments A¹comprises a biologically active moiety, i.e., a moiety that is capableof causing a biological effect when contacted with a cell oradministered to a subject. In some embodiments A¹ comprises or isattached to the TRS via a linker. In some embodiments a linker comprisesa cleavage site, thereby allowing release of at least a portion of A¹when the cleavage site is cleaved, e.g., by a protease in vivo afteradministration of a sortagged cell to a subject. In some embodimentcleavage releases an agent that comprises both a therapeutically activeor detectable moiety and a targeting moiety. The targeting moiety maytarget the released agent to a target cell or site in the body of asubject. Cleavage may occur over a selected time frame so that agent isreleased over a period of time, e.g., to maintain a therapeuticallyuseful level of agent over a period of time. In some embodiments theperiod of time is between 12 and 24 hours, 24 and 48 hours, 2-6 days, orup to about 1, 2, 4, 6, 10, or 12 weeks, or more.

In some aspects, the invention provides methods of using livingmammalian cells that have an agent conjugated thereto via asortase-catalyzed transacylation reaction (sortagged living mammaliancells). Sortagged mammalian cells may be used in vitro, in vivo, inresearch, for detection, for diagnosis, or for therapy. Certain uses ofinterest are described further below but it should be understood thatthe invention is not limited in this respect. Exemplary therapeuticapplications include treatment of infectious diseases, cancer,autoimmune diseases, inflammatory conditions, enzyme deficiencies, orimmunodeficiencies. In some embodiments sortagged living mammalian cellsare used in cell therapy, e.g., in regenerative medicine, adoptiveimmunotherapy, or as vaccine components. In some embodiments sortaggedmammalian cells may be used as delivery vehicles e.g., for deliveringdetection agents or therapeutic agents to a subject. In some embodimentsan agent conjugated to mammalian cells comprises a moiety that is usefulin diagnosis, monitoring, or treatment of a disease. In some embodimentssortagged mammalian cells are administered to a subject, e.g., a subjectin need of diagnosis, monitoring, or treatment of a disease. In someembodiments the cells originate from a subject to whom they aresubsequently administered or originate from a donor who ishistocompatible with the subject.

In some aspects, the invention provides a method of increasing thecirculation time or plasma half-life of an agent in the body of amammal, the method comprising: providing an agent; and conjugating theagent to a mammalian cell using sortase. In some embodiments the methodfurther comprises administering the mammalian cell to the animal, e.g.,directly into the circulatory system, e.g., intravenously. In someembodiments cells may be administered locally, e.g., into a tissue ororgan at which an effect, e.g., a therapeutic effect, is desired. Insome embodiments conjugating an agent to a cell, e.g., a hematologiccell, e.g., a red blood cell, lymphocyte, or red blood cell orlymphocyte precursor, may reduce clearance of the agent, e.g., by thekidneys and/or may reduce diffusion or transport of the agent out of thecirculatory system as compared with the rate at which the unconjugatedagent would be cleared by the kidneys or otherwise removed from thecirculatory system. In some embodiments the mammalian cell or anancestor thereof is obtained from a mammal to whom the sortase-modifiedcell is administered. In some embodiments the average circulation timeor plasma half-life may be increased by at least a factor of 2, 3, 5,10, 20, 50, or more. In some embodiments the average circulation time orplasma half-life may be at least 5, 10, 15, 20, 25, 50 days, or more,e.g., up to the average lifespan of the cell to which the agent isattached.

In some embodiments a therapeutic function may be provided by atherapeutic agent, e.g., an enzyme or therapeutic antibody or smallmolecule, conjugated to a mammalian cell. In some embodiments atherapeutic function may be provided by a moiety targeting a specificcell or cell type to a target site, attracting a specific cell or celltype to a target site, activating a specific cell or cell type, e.g., ata target site, stimulating or inhibiting one or more biologicalactivities of a specific cell or cell type, e.g., at a target site,providing a catalytic activity, e.g., at a target site, or by atherapeutic agent acting on cells, e.g., at a target site. In someembodiments a protein or other agent conjugated to mammalian cellscomprises a binding domain, e.g., an antigen binding domain, or antibodytargeting a specific cell, cell type, tissue, or site, for example, in asubject. In some embodiments the binding domain or antibody isconjugated to a therapeutic agent, for example, a small molecule, or atherapeutic polypeptide. In some embodiments the binding domain orantibody is conjugated to a label. In some embodiments it iscontemplated to attach any therapeutic agent to mammalian cells usingsortase, e.g., any therapeutic agent known in the art.

In some embodiments a mammalian cell has an agent comprising a labelconjugated thereto, e.g., a fluorophore, fluorescent polypeptide,quantum dot, metal-containing nanoparticle, or any other suitable label.The mammalian cell may be detected by detecting the label. In someembodiments the cell may be detected in vitro, e.g., in a cell culturesystem. In some embodiments the cell may be administered to a subjectand detected in vivo. In some embodiments the cell may be administeredto a subject and detected in a sample subsequently obtained from thesubject. In some embodiments the cell has a targeting moiety conjugatedthereto. The targeting moiety and label may be conjugated separately tothe cell or may be part of a single agent conjugated to the cell. Thecells may accumulate at a target site and may be detected in vivo bydetecting the label. In some embodiments the cell may further have atherapeutic agent conjugated thereto or may provide a therapeuticfunction, e.g., a cytotoxic effect against tumor cells or infectedcells.

In some embodiments a mammalian cell is sortagged with a bifunctionalagent, e.g., a bifunctional protein. In some embodiments a bifunctionalagent comprises a first domain that provides a first function and asecond domain that provides a second function. The two domains and/orfunctions may be the same or different. In some embodiments at least onedomain comprises a binding domain that targets the bifunctional agent toa target. A target may be, e.g., an organ, a cell or cell type (e.g., adiseased cell, such as a tumor cell or infected cell), a tissue, or asite of disease). In some embodiments at least one domain provides atherapeutic function or labeling function. Any of a wide variety ofbifunctional agents may be used. In some embodiments a bifunctionalagent comprises a bivalent agent, e.g., a bivalent antibody. A bivalentagent is capable of binding to two molecules or entities, which may bethe same or different, depending on the agent.

In some embodiments a bifunctional agent is a bispecific agent, e.g., abispecific protein, e.g., a bispecific antibody. In some embodiments, abispecific agent targets a specific antigen, cell, cell type, or site ina cell population, tissue, organism, or subject. For example, in someembodiments, a bispecific protein comprises a first binding domain,e.g., an antigen binding domain, that targets the protein to a targetsite (e.g., an organ, a cell or cell type (e.g., a diseased cell, suchas a tumor cell), a tissue, or a site of disease) and a second bindingdomain, e.g., a second antigen binding domain, that provides a function,e.g., a therapeutic function. In some embodiments, a protein or bindingdomain or binding agent binds to a target antigen, e.g., a tumor antigenor an antigen of a pathogen. In some embodiments a binding domain isconjugated to a therapeutic agent, for example, a small molecule, or atherapeutic polypeptide. In some embodiments such conjugation isperformed using click chemistry. For example, sortase may be used toproduce a bifunctional, bivalent, or bispecific agent by installingclick chemistry handles on each of two polypeptides (e.g., scFvs and/orsdAbs) or other molecules, which are then conjugated to each other via aclick chemistry reaction (e.g., as discussed further below). A sortaserecognition motif may be included at or near a free C-terminus and/or a(G)_(n) or (A)_(n) sequence may be included at or near a free N-terminusto facilitate additional sortase-catalyzed conjugation of the agent.

In certain embodiments an agent is a trifunctional agent. Atrifunctional agent may be a trivalent agent, e.g., a trispecific agent.Trivalent agents or agents of even higher valency may be produced assingle polypeptides comprising three or more scFv, sdAb, or acombination thereof. By including scFv and/or or sdAb with differentspecificities in a single polypeptide chain, multispecific (e.g.,bispecific, trispecific) agents are produced. Such agents may havemultiple distinct functions conferred by binding to different moleculesor entities. Sortase may be used to produce trifunctional agents asdescribed for bifunctional agents. Other methods of producingabifunctional or trifunctional agent may also be used. For example,chemical conjugation may be performed using any of a variety ofdifferent approaches (see, e.g., Hermanson, G, cited above).

In some embodiments a particle is conjugated to mammalian cells usingsortase. In some embodiments the particle comprises a detectable labelor therapeutic agent. In some embodiments the particle is a polymericparticle. A detectable label or therapeutic agent may be encapsulatedin, impregnated into, or coated on at least a portion of the surface ofthe particle or otherwise physically associated with the particle. Insome embodiments the label or agent is released from particles over aperiod of time, e.g., to maintain a therapeutically useful level ofagent over a period of time. In some embodiments the period of time isbetween 12 and 24 hours, 24 and 48 hours, 2-6 days, or up to about 1, 2,4, 6, 10, or 12 weeks, or more.

In some embodiments the particle is an ultrasound microbubble orcomprises a contrast agent. In some embodiments the particle has adiameter or average longest axis or length of at least 5 nm, up to about100 nm, 500 nm, 1 μm, 2 μm, or 3 μm. In some embodiments sortagging maybe used to attach a mammalian cell to a support. The support may have anagent comprising a TRS attached thereto, e.g., attached directly to thesupport or to a coating on all or part of the support. The support iscontacted with mammalian cells and sortase under conditions such thatthe agent is conjugated to the cells, thereby attaching the cells to thesupport.

Those of ordinary skill in the art will understand that sortasesubstrates, sortagged mammalian polypeptides, and sortagged cells maycomprise any agent, e.g., any binding agent, therapeutic agent, ordetection agent, that either comprises or can be linked to a polypeptidecomprising a sortase recognition sequence. In some aspects, theinvention encompasses mammalian cells produced according to methodsdescribed herein, and compositions comprising such cells, wherein thecells may be of any cell type and may have any agent conjugated theretousing sortase. In some aspects, the invention encompasses methods ofusing such cells, e.g., for one or more purposes described herein.

In some embodiments a mammalian cell is sortagged with an agent that iscapable of binding to one or more entities. Such a moiety may bereferred to as a “binding moiety”. In some embodiments an agentcomprising a binding moiety is attached to a mammalian cell as describedherein, and the mammalian cell is placed in an environment comprisingone or more entities. The binding moiety causes the mammalian cell tobecome attached (bound) to at least one of the entities via interactionbetween the binding moiety and the entity. The entity may contain aspecific domain, moiety, or binding site that physically interacts withthe binding moiety. In general a binding moiety may be any moietycapable of specifically recognizing an entity of interest. It will beunderstood that a binding moiety may recognize only a portion of anentity, e.g., an epitope of a protein. In general, a binding moiety thatbinds to a particular entity may be any moiety capable of formingappropriate interactions with the entity. In some embodiments a bindingmoiety may be a protein, a peptide, an antibody, an antibody fragment,an engineered binding protein (e.g., an affibody, anticalin, oradnectin), a nucleic acid aptamer, a naturally occurring or artificialligand, etc. In some embodiments a ligand is a small molecule. Forexample, a binding moiety may be a small molecule that binds to areceptor. In some embodiments a binding moiety may be a receptor, whichmay bind to an entity comprising a ligand of the receptor. In certainembodiments two, three, or more binding moieties with the same ordifferent specifities may be combined (e.g., by chemical linkage,production as a fusion protein, or by sortase-catalyzed reactions) toform a multivalent agent, e.g., a bivalent or trivalent agent. In someembodiments an agent comprises a multimer or concatamer comprising 3-10,10-25, 25-50, 50-100, 100-1000 binding moieties, or more. In someembodiments, a multivalent agent may have higher affinity or avidity fora target than does an agent comprising a single binding moiety.

In some embodiments a binding moiety is capable of targeting an agent toa target of interest. Such a binding moiety may be referred to as a“targeting moiety”. In some embodiments an agent comprising a targetingmoiety is attached to a mammalian cell and targets the mammalian cell toa target of interest. In general, any binding moiety may be used as atargeting moiety, provided that the binding moiety recognizes and bindsto a target of interest. A target of interest may be any of a variety ofdifferent entities. In some embodiments a target of interest isassociated with or comprises a cell, structure, or molecule. In someembodiments a target of interest is a normal cell. In some embodiments atarget of interest is an abnormal cell, e.g., a diseased cell such as acancer cell or infected cell. In some embodiments a targeting moietybinds to a polypeptide, lipid, or sugar exposed at the surface of atarget cell, e.g., an extracellular domain of a polypeptide expressed bythe target cell. In some embodiments a target of interest is orcomprises a specific antigen, cell type, or site in tissue, organ, orsubject. In some embodiments a targeting moiety binds to a marker on acell of interest. In some embodiments a target or marker specific for adiseased cell or site of disease.

In some embodiments a population of mammalian cells is contacted withtwo or more different sortase substrates each comprising a sortaserecognition motif and a different agent, to produce a compositioncomprising cells that are sortagged with at least two different agents.In some embodiments a population of mammalian cells is divided intomultiple aliquots. The number of aliquots and number of cells peraliquot may be selected in any convenient manner. In some embodiments analiquot comprises at least 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ cells. Insome embodiments the number of aliquots is between 2 and 1,000. One ormore aliquots may be stored for future use. In some embodiments two ormore different sortase substrates each comprising a sortase recognitionmotif and a different agent conjugated to the sortase recognition motif,are conjugated to cells of two or more different aliquots, to producetwo or more populations of mammalian cells having different agentsconjugated thereto. The different aliquots, or portions thereof, may besubsequently combined. Different agents may be of the same or differentcompound classes (e.g., polypeptides, polynucleotides, small molecules).Different agents may or may not be related in sequence or structure orcapable of binding to the same target.

In some embodiments two, three, or more sequential sortagging reactionsare performed. In some embodiments, cells are contacted with a sortaseand a first substrate comprising a sortase recognition sequence and afirst agent, and the sortagging reaction is allowed to proceed for atime and under conditions appropriate to sortag the cells with the firstagent. Cells are then separated from the first substrate, e.g., byremoving the cells or the first substrate from the vessel. The sortaggedcells are then contacted with a second substrate comprising a sortaserecognition sequence and a second agent, and the sortagging reaction isallowed to proceed for a time and under conditions appropriate to sortagthe cells with the second agent, resulting in cells that are sortaggedwith the first agent and with a second agent. In some embodiments, cellsare contacted with a sortase and a first substrate comprising a sortaserecognition sequence and a first agent, and the sortagging reaction isallowed to proceed for a time and under conditions appropriate to sortagthe cells with the first agent. A second sortase substrate is then addedto the reaction mixture without separating the first sortase substrate,and the reaction is allowed to proceed. Factors such as the time andconditions of each sortagging reaction, the order in which differentsubstrates are added or used, etc., may be adjusted to achieve a desiredproportion of first and second agents attached to the cells. In someembodiments in which two or more substrates have different molecularweights, a substrate having a higher molecular weight may be used beforea substrate having a lower molecular weight. The process may be repeatedone or more times. If desired, a time course may be conducted to monitorthe extent of sortagging over time. For example, conditions that resultin a reaction that goes a selected portion of the way to completion(maximum conjugation) may be determined (e.g., 25%, 50%, 75%, 90%, ormore). Information obtained from the time course may be used to optimizethe reactions to achieve desired ratio of different agents on the cellsurface. Of course each sortagging reaction may be conducted using amixture of sortase substrates together, thus potentially resulting inany number of different agents, e.g., 3, 4, 5, 6, or more, conjugated tothe cells.

In some embodiments, the total number of molecules of agent(s)conjugated to a mammalian cell using sortase according to the presentinvention is between 10 and 100; between 100 and 1,000; between 1,000and 10,000; between 10,000 and 50,000; between 50,000 and 100,000;between 100,000 and 500,000; between 500,000 and 1,000,000; between1,000,000 and 2,500,000; between 2,500,000 and 5,000,000; between5,000,000 and 10,000,000, or more. In some embodiments, the averagenumber of agent molecules per cell conjugated to mammalian cells in apreparation of mammalian cells sortagged according to the presentinvention is between 10 and 100; between 100 and 1,000; between 1,000and 10,000; between 10,000 and 50,000; between 50,000 and 100,000;between 100,000 and 500,000; between 500,000 and 1,000,000; between1,000,000 and 2,500,000; between 2,500,000 and 5,000,000; between5,000,000 and 10,000,000, or more. The number, or average number, ofmolecules per cell may be controlled, if desired, by appropriateselection of the reaction conditions, e.g., by controlling one or morefactors such as the sortase used, the temperature, and/or the durationof the reaction. The number, or average number, of molecules per cellmay also vary depending on the available surface area or cell type. Insome embodiments the molecules of agent(s) conjugated to a non-mammaliancell using sortase according to the present invention is as mentionedfor mammalian cells. In certain embodiments a cell preparation ischaracterized in that least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more of the cells in the cell preparation have a number of agentmolecules within any of the afore-mentioned ranges conjugated to them.

Without wishing to be bound by any theory, sortase-mediated modificationof mammalian cells that are not genetically engineered may have one ormore advantages for a variety of purposes, e.g., for certain purposes inwhich the cells are administered to subjects. Use of non-geneticallyengineered cells may, for example, permit modification of cells that arerefractory to genetic engineering, avoid the potentially time-consumingstep of genetic engineering, and/or avoid safety concerns that may arisewhen genomic sequence is modified using, e.g., viral vectors such asretroviruses to insert a nucleic acid into the genome. Such concerns mayinclude potential insertional mutagenesis, which may lead to activationof oncogenes or inactivation of tumor suppressor genes.

While use of non-genetically engineered cells may have certainadvantages, unless otherwise indicated or clearly evident from thecontext, any of the methods of generating or using sortase-modifiedanimal cells described herein may, in certain embodiments, use animalcells that have been genetically engineered to express a polypeptidethat comprises a sortase recognition motif or nucleophilic acceptorsequence, so that the polypeptide is suitable for use as a sortasesubstrate or nucleophile in a sortase-mediated reaction. Similarly,unless otherwise indicated or clearly evident from the context, any ofthe compositions comprising sortase-modified animal cells or useful forgenerating or using such cells may, in certain embodiments, use cellsthat have been genetically engineered to express a polypeptide thatcomprises a sortase recognition motif or nucleophilic acceptor sequenceso that the polypeptide is suitable for use as a sortase substrate ornucleophile in a sortase-mediated reaction.

In some embodiments, genetically engineered cells are modified by usingsortase to attach a sortase substrate to a non-genetically engineeredendogenous polypeptide of the cell. The cell may, for example, have beengenetically engineered to express any of a wide variety of products,e.g., polypeptides or noncoding RNAs, may be genetically engineered tohave a deletion of at least a portion of one or more genes, and/or maybe genetically engineered to have one or more precise alterations in thesequence of one or more endogenous genes. In certain embodiments anon-engineered endogenous polypeptide of such genetically engineeredcell is sortagged with any of the various agents described herein.

Although the invention is described herein mainly in regard to mammaliancells, the invention provides embodiments in which any eukaryotic cell,e.g., any animal cells, e.g., any vertebrate cells, e.g., avian cells,fish cells, amphibian cells, or reptilian cells, or invertebrate animalcells, e.g., insect cells, or fungal cells (e.g., yeast), or protozoalcells may be used, in any aspect described herein. Accordingly, wherethe disclosure refers to mammalian cells, it should be understood thatanalogous aspects and embodiments pertaining to other eukaryotic celltypes, e.g., fungal, insect, protozoal, are provided unless otherwiseindicated or evident from the context. In some embodiments polypeptidesendogenous to such cells may be sortagged.

In certain embodiments, sortagging eukaryotic cells, e.g., animal cells,as described herein does not comprise and/or is not performed inconnection with sortagging cells that have been genetically engineeredfor sortagging. For example, the method is not performed as a negativecontrol in connection with sortagging cells that have been geneticallyengineered to comprise a protein comprising a sortase recognitionsequence or nucleophilic acceptor sequence. In certain embodiments themethod is performed in order that the sortagged animal cells that havenot been engineered for sortagging may be used for one or more purposesof interest. In certain embodiments the sortagging occurs at a levelabove what would reasonably be expected as background level ofnonspecific binding of a sortase substrate to an animal cell. In someembodiments the number of sortagged cells produced is sufficient toadminister a therapeutically effective amount of an agent to a mammaliansubject, e.g., a human.

II. Suitable Transamidase Enzymes and Transamidase Recognition Motifs

Enzymes identified as “sortases” have been isolated from a variety ofGram-positive bacteria. In nature, these enzymes catalyze a cell wallsorting reaction in which a surface protein with a sorting signalcontaining a sortase recognition motif is cleaved and the carboxyl endof the protein is covalently attached to a pentaglycine cross-bridge ofpeptidoglycan. Gram-positive bacteria include the following genera:Actinomyces, Bacillus, Bifidobacterium, Cellulomonas, Clostridium,Corynebacterium, Micrococcus, Mycobacterium, Nocardia, Staphylococcus,Streptococcus, and Streptomyces. In certain embodiments thetranspeptidation reaction catalyzed by sortase results in the ligationof species containing a sortase recognition motif with species bearingone or more N-terminal glycine residues or an N-terminal alkylaminegroup. Sortases, sortase-mediated transacylation reactions, and theiruse in protein engineering are well known to those of ordinary skill inthe art (see, e.g., Ploegh et al., International Patent ApplicationsPCT/US2010/000274 (WO/2010/087994), and PCT/US2011/033303(WO/2011/133704).

Additional description of use of sortase, sortase preparation methods,sortases, sortase substrates, sortase recognition sequences, etc., maybe found in Popp M W, Ploegh H L., Angew Chem Int Ed Engl, 2011;50:5024-5032; Strijbis, K., et al., Traffic 2012; 13: 780-789; Witte MD, et al., Proc Natl Acad Sci USA. 2012; 109(30):11993-8; Hess G T, etal., Bioconjug Chem. 2012 Jul. 18; 23(7):1478-87, Witte M D et al.(2012) PNAS 109:11993-11998; Guimaraes C P et al. (2013) Site-specificC-terminal and internal loop labeling of proteins using sortase-mediatedreactions. Nat Protoc 8:1787-1799, and references in any of these.

Sortases have been classified into 4 classes, designated A, B, C, and D,based on sequence alignment and phylogenetic analysis of 61 sortasesfrom Gram positive bacterial genomes (Dramsi S, Trieu-Cuot P, Bierne H,Sorting sortases: a nomenclature proposal for the various sortases ofGram-positive bacteria. Res Microbiol. 156(3):289-97, 2005. Theseclasses correspond to the following subfamilies, into which sortaseshave also been classified by Comfort and Clubb (Comfort D, Clubb R T. Acomparative genome analysis identifies distinct sorting pathways ingram-positive bacteria. Infect Immun., 72(5):2710-22, 2004): Class A(Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3), Class D(Subfamilies 4 and 5). The aforementioned references disclose numeroussortases and recognition motifs. See also Pallen, M. J.; Lam, A. C.;Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101.Those skilled in the art will readily be able to assign a sortase to thecorrect class based on its sequence and/or other characteristics such asthose described in Drami, et al., supra. The term “sortase A” is usedherein to refer to a class A sortase, usually named SrtA in anyparticular bacterial species, e.g., SrtA from S. aureus or S. pyogenesLikewise “sortase B” is used herein to refer to a class B sortase,usually named SrtB in any particular bacterial species, e.g., SrtB fromS. aureus. The present disclosure encompasses embodiments relating toany of the sortase classes known in the art (e.g., a sortase A from anybacterial species or strain, a sortase B from any bacterial species orstrain, a class C sortase from any bacterial species or strain, and aclass D sortase from any bacterial species or strain). In certainembodiments a sortase that utilizes a nucleophilic acceptor sequencehaving an N-terminal glycine, e.g., 1-5 N-terminal glycines, is used,such as SrtA from S. aureus. In some embodiments it is contemplated touse two or more sortases. In some embodiments the sortases may utilizedifferent sortase recognition sequences and/or different nucleophilicacceptor sequences. For example, SrtA from S. pyogenes can utilize anucleophilic acceptor sequence having one or more N-terminal alanines,e.g., 1-5 N-terminal alanines and/or may utilize a sortase recognitionmotif comprising LPXTA.

Amino acid sequences of Srt A and Srt B and the nucleotide sequencesthat encode them are known to those of skill in the art and aredisclosed in a number of references cited herein, the entire contents ofall of which are incorporated herein by reference. The amino acidsequences of S. aureus SrtA and SrtB are homologous, sharing, forexample, 22% sequence identity and 37% sequence similarity. The aminoacid sequence of a sortase-transamidase from Staphylococcus aureus alsohas substantial homology with sequences of enzymes from otherGram-positive bacteria, and such transamidases can be utilized in theligation processes described herein. For example, for SrtA there isabout a 31% sequence identity (and about 44% sequence similarity) withbest alignment over the entire sequenced region of the S. pyogenes openreading frame. There is about a 28% sequence identity with bestalignment over the entire sequenced region of the A. naeslundii openreading frame. It will be appreciated that different bacterial strainsmay exhibit differences in sequence of a particular polypeptide, and thesequences herein are exemplary.

In certain embodiments a transamidase bearing 18% or more sequenceidentity, 20% or more sequence identity, or 30% or more sequenceidentity with the S. aureus, S. pyogenes, A. naeslundii, S. nutans, E.faecalis or B. subtilis open reading frame encoding a sortase can bescreened, and enzymes having transamidase activity comparable to Srt Aor Srt B from S. aureas can be utilized (e. g., comparable activitysometimes is 10% of Srt A or Srt B activity or more).

Thus in some embodiments of the invention the sortase is a sortase A(SrtA). SrtA recognizes the motif LPXTG, with common recognition motifsbeing, e.g., LPKTG, LPATG, LPNTG. In some embodiments LPETG is used.However, motifs falling outside this consensus may also be recognized.For example, in some embodiments the motif comprises an ‘A’ rather thana ‘T’ at position 4, e.g., LPXAG, e.g., LPNAG. In some embodiments themotif comprises an ‘A’ rather than a ‘G’ at position 5, e.g., LPXTA,e.g., LPNTA. In some embodiments the motif comprises a ‘G’ rather than‘P’ at position 2, e.g., LGXTG, e.g., LGATG. In some embodiments themotif comprises an ‘I’ rather than ‘L’ at position 1, e.g., IPXTG, e.g.,IPNTG or IPETG.

In some embodiments, a variant of a naturally occurring sortase may beused. Such variants may be produced through processes such as directedevolution, site-specific modification, etc. Considerable structuralinformation regarding sortase enzymes, e.g., sortase A enzymes, isavailable, including NMR or crystal structures of SrtA alone or bound toa sortase recognition sequence (see, e.g., Zong Y, et al. J. Biol Chem.2004, 279, 31383-31389). Three dimensional structure information is alsoavailable for other sortases, e.g., S. pyogenes SrtA (Race, P R, et al.,J Biol Chem. 2009, 284(11):6924-33). The active site and substratebinding pocket of S. aureus SrtA have been identified. One of ordinaryskill in the art can generate functional variants by, for example,avoiding deletions or substitutions that would disrupt or substantiallyalter the active site or substrate binding pocket of a sortase. In someembodiments a functional variant of S. aureus SrtA comprises His atposition 120, Cys at position 184, and Arg at position 197, wherein Cysat position 184 is located within a TLXTC motif. Functional variants ofother SrtA proteins may have His, Cys, Arg, and TLXTC motifs atpositions that correspond to the positions of these residues in S.aureus SrtA. In some embodiments, a sortase variant comprises a sequenceat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a wild type sortase A sequence or catalytic domain thereof,e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to amino acids 60-206 of SEQ ID NO: 1 or SEQ ID NO: 2, orat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to amino acids 26-206 of SEQ ID NO: 1 or SEQ ID NO: 2. In someembodiments, a sortase variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 amino acid substitutions relative to amino acids60-206 of SEQ ID NO: 1 or relative to amino acids 26-206 of SEQ ID NO: 1or SEQ ID NO: 2.

In some embodiments, a transamidase having higher transamidase activitythan a naturally occurring sortase may be used. In some embodiments theactivity of the transamidase is at least about 10, 15, 20, 40, 60, 80,100, 120, 140, 160, 180, or 200 times as high as that of S. aureussortase A. In some embodiments the activity is between about 10 and 50times as high as that of S. aureus sortase A, e.g., between about 10 and20 times as high, between about 20 and 30 times as high, between about30 and 50 times as high. In some embodiments the activity is betweenabout 50 and about 150 times as high as that of S. aureus sortase A,e.g., between about 50 and 75 times as high, between about 75 and 100times as high, between about 100-125 times as high, or between about 125and 150 times as high. For example, variants of S. aureus sortase A withup to a 140-fold increase in LPETG-coupling activity compared with thestarting wild-type enzyme have been identified (Chen, I., et al., PNAS108(28): 11399-11404, 2011). In some embodiments such a sortase variantis used in a composition or method of the invention. In some embodimentsa sortase variant comprises any one or more of the followingsubstitutions relative to a wild type S. aureus SrtA: P94S or P94R,D160N, D165A, K190E, and K196T mutations.

One of ordinary skill in the art will appreciate that the foregoingdescriptions of substitutions utilize standard notation of the formX₁NX₂, in which X₁ and X₂, represent amino acids and N represents anamino acid position, X₁ represents an amino acid present in a firstsequence (e.g., a wild type S. aureus SrtA sequence), and X₂ representsan amino acid that is substituted for X₁ at position N, resulting in asecond sequence that has X₂ at position N instead of X₁. It should beunderstood that the present disclosure is not intended to be limited inany way by the identity of the original amino acid residue X₁ that ispresent at a particular position N in a wild type SrtA sequence used togenerate a SrtA variant and is replaced by X₂ in the variant. Anysubstitution which results in the specified amino acid residue at aposition specified herein is contemplated by the disclosure. Thus asubstitution may be defined by the position and the identity of X₂,whereas X₁ may vary depending, e.g., on the particular bacterial speciesor strain from which a particular SrtA originates. Thus in someembodiments, a sortase A variant comprises any one or more of thefollowing: an S residue at position 94 (S94) or an R residue at position94 (R94), an N residue at position 160 (N160), an A residue at position165 (A165), an E residue at position 190 (E190), a T residue at position196 (T196) (numbered according to the numbering of a wild type SrtA,e.g., SEQ ID NO: 1). For example, in some embodiments a sortase Avariant comprises two, three, four, or five of the afore-mentionedmutations relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1).In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94), and also an Nresidue at position 160 (N160), an A residue at position 165 (A165), anda T residue at position 196 (T196). For example, in some embodiments asortase A variant comprises P94S or P94R, and also D160N, D165A, andK196T. In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94) and also an Nresidue at position 160 (N160), A residue at position 165 (A165), a Eresidue at position 190, and a T residue at position 196. For example,in some embodiments a sortase A variant comprises P94S or P94R, and alsoD160N, D165A, K190E, and K196T. In some embodiments a sortase A variantcomprises an R residue at position 94 (R94), an N residue at position160 (N160), a A residue at position 165 (A165), E residue at position190, and a T residue at position 196. In some embodiments a sortasecomprises P94R, D160N, D165A, K190E, and K196T.

It is to be further understood that the disclosure contemplates variantsof any wild-type sortase A. Those skilled in the art will appreciatethat wild-type sequences of sortase A may vary, e.g., SrtA from variousspecies may have gaps, insertions, and/or may vary in length relative tothe amino acid sequence of exemplary wild-type S. aureus SrtA. Thoseskilled in the art will appreciate that the positions described hereinin regard to substitutions or other alterations pertain to the sequenceof exemplary wild type S. aureus SrtA, unless otherwise indicated, andthat such positions may be adjusted when making correspondingsubstitutions in different bacterial SrtA sequences in order to accountfor such gaps, insertions, and/or length differences. For example, asnoted above, certain sortase variants comprise a substitution at aminoacid position 94 (e.g., the amino acid is changed to an S residue).However, the amino acid at position 94 in S. aureus SrtA may correspondto an amino acid at a different position (e.g., position Z) in SrtA froma second bacterial species when the sequences are aligned. Whengenerating a variant of the SrtA of the second bacterial speciescomprising a substitution at “position 94” (based on the wild type S.aureus SrtA sequence numbering), it is the amino acid at position Z ofthe SrtA from the second bacterial species that should be changed (e.g.,to S) rather than the amino acid at position 94. Those skilled in theart will understand how to align any original wild-type sortase Asequence to be used for generating a SrtA variant with an exemplarywild-type S. aureus sortase A sequence for purposes of determining thepositions in the original wild-type sortase A sequence that correspondto the exemplary wild-type S. aureus sortase A sequence when taking intoaccount gaps and/or insertions in the alignment of the two sequences.

In some embodiments, amino acids at position 94, 160, 165, 190, and/or196 are altered in a variant as compared with the amino acids present atthose positions in a wild type S. aureus SrtA, and the other amino acidsof the variant are identical to those present at the correspondingpositions in a wild type SrtA, e.g., a wild type S. aureus SrtA. In someembodiments, one or more of the other amino acids of a variant, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 of the other amino acids differ from thosepresent at corresponding position(s) in a wild type SrtA, e.g., a wildtype S. aureus SrtA. In some embodiments a variant may have any of theproperties or degrees of sequence identity specified in the definitionof “variants” above.

An exemplary wild type S. aureus SrtA sequence (Gene ID: 1125243, NCBIRefSeq Acc. No. NP_375640.1) is shown below, with the afore-mentionedpositions underlined:

(SEQ ID NO: 1) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDNKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK.One of ordinary skill in the art will appreciate that differentsubspecies, strains, and isolates may differ in sequence at positionsthat do not significantly affect activity. For example, anotherexemplary wild type S. aureus SrtA sequence (Gene ID: 3238307, NCBIRefSeq Acc. No. YP_187332.1; GenBank Acc. No. AAD48437) has a K residueat position 57 and a G residue at position 167, as shown below in SEQ IDNO: 2:

(SEQ ID NO: 2) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK

Either or both of these amino acids (i.e., K57 and/or G167) may bepresent in or introduced into any SrtA sequence, e.g., any S. aureusSrtA sequence, whether naturally occurring or generated by man.Furthermore, as described herein, any sortase sequence may furthercomprise a tag (e.g., 6×His), a spacer, or both. For example, the N- orC-terminus may be extended to encompass a tag, optionally separated fromthe rest of the sequence by a spacer,

In some embodiments a sortase variant comprising the following sequencemay be used, in which amino acid substitutions relative to a wild typeS. aureus SrtA of SEQ ID NO: 1 or SEQ ID NO: 2 are shown in underlinedbold letters:

(SEQ ID NO: 3) MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPAT R EQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI R N VKPT AVEVLDEQKGKDKQLTLITCDDYNE E TGVWE T RKIFVATEVK.

As will be appreciated, amino acids 2-148 of the above sequencecorrespond to amino acids 60-206 of the full length S. aureus SrtAsequence (the catalytic domain). For example, the “R” residue atposition 36 of SEQ ID NO: 3 corresponds to the “P” residue at position94 in SEQ ID NO: 1 or 2. It is also contemplated in some embodiments touse sortase variants that have other substitutions at one or more ofpositions 94, 160, 165, 190, and 196 (numbered according to thenumbering of SEQ ID NO: 1 or 2), e.g., wherein such substitutionsutilize an amino acid that would be a conservative substitution at therelevant position as compared with the sequence of SEQ ID NO: 3.

In some embodiments a calcium-independent sortase, e.g., acalcium-independent sortase A, is used. In some embodiments acalcium-independent variant of S. aureus SrtA is used. As used herein“calcium-independent” refers to the ability of a sortase enzyme, e.g., asortase A enzyme, to exhibit catalytic activity in a manner that issubstantially independent of the absence, presence, or concentration ofcalcium, at least across a concentration range of about 0 mM-about 10mM. For example, in some embodiments the activity of acalcium-independent sortase in an aqueous medium that comprises acalcium chelator such as EDTA or EGTA at a concentration sufficient tochelate substantially all calcium ions is equal to or at leastapproximately 80%, 85%, 90%, 95%, or more as great as its activity inthe same medium in the absence of the calcium chelator. Acalcium-independent sortase may exhibit calcium-independent activity athigher calcium concentrations as well, e.g., up to any concentrationthat is not detrimental to proper functioning of the enzyme. In someembodiments, a sortase can be assayed for ability to exhibit sortasecatalytic activity in the presence of calcium concentrations that arelower than calcium concentrations which are required forcalcium-dependent sortase to exhibit catalytic activity. As used herein,“calcium-dependent” in connection with a sortase means that thecatalytic activity of the sortase relies or depends on the presence andconcentration of calcium, such that in the absence of calcium or theabsence of a sufficient amount of calcium, the calcium-dependent sortasewill not exhibit sortase catalytic activity or has greatly reducedcatalytic activity (e.g., less than about 5%, or less than about 10%, ofthe activity that it has when calcium is present in sufficient amounts(e.g., 5 mM-10 mM)). In some embodiments a calcium-dependent sortase,e.g., a wild type S. aureus SrtA or catalytic domain thereof, is used tosortag eukaryotic cells in a medium containing more than 0.5 mM calcium,e.g., at least 1.0 mM, at least 2.0 mM, at least 3.0 mM, at least 4.0mM, or at least 5.0 mM calcium. For example, the concentration ofcalcium may be 1.0 mm-2.5 mM, 2.5 mM-5.0 mM, 5.0 mM-7.5 mM, 7.5 mM-10.0mM, 10.0 mM-15 mM, 15 mM-20 mM. In some embodiments, acalcium-independent sortase may be used to sortag eukaryotic cells in amedium that lacks calcium or has a low calcium concentration (e.g., acalcium concentration below that at which the sortase exhibits maximumactivity).

A sortase (e.g., a sortase having a naturally occurring sortase sequenceor a sortase variant generated by man) can be assayed for ability toexhibit sortase catalytic activity in a calcium-independent manner by,for example, contacting a target protein comprising a C-terminal sortaserecognition motif with a tagged N-terminal oligoglycine derivative inthe absence of calcium in the presence of the sortase and determiningwhether the target protein is ligated to the tagged N-terminaloligoglycine derivative by the sortase. In some embodiments, catalyticactivity may be measured by the yield of sortagged target protein aftera selected time period, e.g., about 6, 12, or 18 hours of reaction. Insome embodiments, catalytic activity may be measured by measuringk_(cat), K_(m), and/or k_(cat)/K_(m). In some embodiments, one or morekinetic parameters of SrtA activity (e.g., k_(cat), k_(cat)/K_(m)) maybe determined as described in Ton-That et al., J Biol Chem. 2000;275(13):9876-81. In some embodiments a calcium-independent sortase is avariant of a calcium-dependent sortase, wherein the variant comprisesone or more amino acid substitutions relative to the calcium-dependentsortase. In some embodiments a calcium-independent variant has a k_(cat)at least about 25%, 30%, 40%, 45%, 50%, 55%, 60%, or more as high asthat of a calcium-dependent sortase of which it is a variant. In someembodiments a calcium-independent variant has a k_(cat)/K_(m) at leastabout 25%, 30%, 40%, 45%, 50%, 55%, 60%, or more as high as that of acalcium-dependent sortase of which it is a variant.

In some embodiments a calcium-independent sortase is a naturallyoccurring sortase, e.g., SrtA from S. pyogenes B. anthracis, E.faecalis, L. plantarum, L. lactis, or L. monocytogenes. In someembodiments a calcium-independent sortase is a S. aureus SrtA variant.In some embodiments, the present invention contemplates use of anysortase A, e.g., any mutant of an S. aureus SrtA, described inco-pending US provisional patent application entitled“Calcium-Independent Sortase A Mutants”, Attorney Docket Number:WIBR-141-001, filed on even date herewith, which is hereby incorporatedby reference. It should be noted that the term “mutant” is usedinterchangeably with “variant” and should not be considered to implythat any particular way of generating the mutant sequences is requiredor that any particular starting materials is required. The disclosurecontemplates any suitable method of generating variants. Examples ofsuitable methods include, but are not limited to, introducing mutationsinto an appropriate wild-type coding sequence (e.g., using site-specificmutagenesis), synthesizing the sequences of the variants de novo, forexample, utilizing solid phase peptide synthesis, and in vitrotranslation a synthetic mRNA, to name only a few. Calcium-independentsortases may be used in various embodiments of any method or compositiondescribed herein. In some embodiments, a calcium-independent S. aureusSrtA variant has a mutation at position 105 and position 108 as comparedwith a wild type S. aureus SrtA. For example, glutamine (E) residues atposition 105 and position 108 in a wild type S. aureus SrtA sequence maybe changed to a residue that is present at the corresponding position ina calcium-independent sortase (e.g., K or Q, respectively). In someembodiments, for example, a sortase variant comprises an E105K and anE108A substitution or an E105K and an E108Q substitution in a wild typeS. aureus SrtA sequence or functional variant or fragment thereof.

In some embodiments, a calcium-independent sortase A variant comprisesat least three amino acid substitutions relative to a wild-type sortaseA, wherein the amino acid substitutions comprise a) a K residue atposition 105; b) a Q or A residue at position 108; and c) at least oneamino acid substitution selected from the group consisting of i) a Rresidue at position 94; ii) a S residue at position 94; iii) a N residueat position 160; iv) a A residue at position 165; v) a E residue atposition 190; and vi) a T residue at position 196. In some embodiments acalcium-independent sortase A variant comprises the following amino acidsubstitutions relative to a wild-type sortase A: a) a K residue atposition 105; b) a Q or A residue at position 108; c) an S residue atposition 94 or R residue at position 94; d) an N residue at position160; e) an A residue at position 165, and a T residue at position 196.In some embodiments, a calcium-independent sortase A variant comprisesthe following amino acid substitutions relative to a wild-type sortaseA: a) a K residue at position 105; b) a Q or A residue at position 108;c) a R or S residue at position 94; d) a N residue at position 160; e) aA residue at position 165; f) a E residue at position 190; and g) a Tresidue at position 196. In some embodiments a sortase comprises thefollowing sequence, in which amino acids at positions 94, 105, 108, 160,165, 190, and 196 relative to a full length S. aureus SrtA sequence areshown in bold:

(SEQ ID NO: 4) MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQSLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVK.

In some embodiments a transamidase that has an altered substrateselectivity as compared with a naturally occurring sortase may be used.For example, variants of S. aureus sortase A that accept aromatic aminoacids (e.g., phenylalanine), as well as amino acids with small sidechains such as Ala, Asp, Ser, Pro, and Gly, at position 1 of the sortaserecognition motif (instead of L) have been identified (Piotukh K, etal., J Am Chem Soc., 133(44):17536-9, 2011). In some embodiments such asortase is used in a composition or method of the invention. A sortasewith an altered substrate selectivity with regard to the sortaserecognition motif may be generated by engineering one or more mutationsin the sortase, e.g., in a region of the protein that is involved inrecognition and/or binding of the sortase recognition motif, e.g., theputative substrate recognition loop (e.g., the loop connecting strandsβ6 and β7 (β6/β7 loop) in SrtA (Val161-Asp176). A crystal structure ofS. aureus SrtA and a substrate, illustrating the loops, is described inZong, Y., et al., J Biol Chem. 2004 Jul. 23; 279(30):31383-9.). In someembodiments, a phage-display, yeast display, or other screen of a mutantsortase library randomized in the substrate recognition loop may beperformed, and variants with altered substrate specificity may beidentified.

In some embodiments the sortase is a sortase B (SrtB), e.g., a sortase Bof S. aureus, B. anthracis, or L. monocytogenes. Motifs recognized bysortases of the B class (SrtB) often fall within the consensus sequencesNPXTX, e.g., NP[Q/K]-[T/s]-[N/G/s], such as NPQTN or NPKTG. For example,sortase B of S. aureus or B. anthracis cleaves the NPQTN or NPKTG motifof IsdC in the respective bacteria (see, e.g., Marraffini, L. andSchneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007).Other recognition motifs found in putative substrates of class Bsortases are NSKTA, NPQTG, NAKTN, and NPQSS. For example, SrtB from L.monocytogenes recognizes certain motifs lacking P at position 2 and/orlacking Q or K at position 3, such as NAKTN and NPQSS (Mariscotti J F,et al., The Listeria monocytogenes sortase-B recognizes varied aminoacids at position two of the sorting motif. J Biol Chem. 2009 Jan. 7.[Epub ahead of print]).

In some embodiments, the sortase is a class C sortase. Class C sortasesmay utilize LPXTG as a recognition motif.

In some embodiments, the sortase is a class D sortase. Sortases in thisclass are predicted to recognize motifs with a consensus sequenceNA-[E/A/S/H]-TG (Comfort D, supra). Class D sortases have been found,e.g., in Streptomyces spp., Corynebacterium spp., Tropheryma whipplei,Thermobifida fusca, and Bifidobacterium longhum. LPXTA or LAXTG mayserve as a recognition sequence for class D sortases, e.g., ofsubfamilies 4 and 5, respectively subfamily-4 and subfamily-5 enzymesprocess the motifs LPXTA and LAXTG, respectively). For example, B.anthracis Sortase C, which is a class D sortase, has been shown tospecifically cleave the LPNTA motif in B. anthracis BasI and BasH(Marrafini, supra).

See Barnett and Scott for description of a sortase from that recognizesQVPTGV motif (Barnett, T C and Scott, J R, Differential Recognition ofSurface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs.Journal of Bacteriology, Vol. 184, No. 8, p. 2181-2191, 2002).

The invention contemplates use of sortases found in any gram positiveorganism, such as those mentioned herein and/or in the references(including databases) cited herein. The invention also contemplates useof sortases found in gram negative bacteria, e.g., Colwelliapsychrerythraea, Microbulbifer degradans, Bradyrhizobium japonicum,Shewanella oneidensis, and Shewanella putrefaciens. They recognizesequence motifs LP[Q/K]T[A/S]T. In keeping with the variation toleratedat position 3 in sortases from gram positive organisms, a sequence motifLPXT[A/S], e.g., LPXTA or LPSTS may be used. Use of sortases fromArchaea (e.g. Methanobacterium thermoautotrophicum) is contemplated incertain embodiments.

In some embodiments, the sortase, or transamidase, recognition sequenceis LPXTG, wherein X is a standard or non-standard amino acid. In someembodiments, X is selected from D, E, A, N, Q, K, or R. In someembodiments, the recognition sequence is selected from LPXTG, SPXTG,LAXTG, LSXTG, NPXTG, VPXTG, IPXTG, and YPXRG, wherein X may be selectedfrom D, E, A, N, Q, K, or R in certain embodiments. In some embodimentsa C-terminal G is replaced by A. In some embodiments X is selected tomatch a naturally occurring transamidase recognition sequence.

In certain embodiments the C-terminal amino acid residue of a sortaserecognition motif may be replaced with a moiety that exhibits poorernucleophilicity once released from the sortase (PCT/US2010/000274;Antos, J., et al., J. Am. Chem. Soc., 2009, 131 (31), pp 10800-10801).For example, the G in LPXTG may be replaced by a moiety that exhibitspoorer nucleophilicity than glycine once released from the sortase, suchas an alkyl ester, e.g., a methyl ester. In some embodiments, thetransamidase recognition sequence is selected from: LPKT, LPIT, LPDT,SPKT, LAET, LAAT, LAET, LAST, LAET, LPLT, LSRT, LPET, VPDT, IPQT, YPRR,LPMT, LPLT, LAFT, LPQT, NSKT, NPQT, NAKT, and NPQS. In certainembodiments any of the afore-mentioned TRSs having four amino acids mayfurther comprise a moiety that exhibits a pooer nucleophilicity thanglycine once released from the sortase.

In some embodiments, e.g., in certain embodiments in which sortase A isused, the transamidase recognition motif comprises the amino acidsequence X₁PX₂X₃ or X₁PX₂X₃G, where X₁ is leucine, isoleucine, valine ormethionine; X₂ is any amino acid; X₃ is threonine, serine or alanine; Pis proline and G is glycine. In specific embodiments, as noted above X₁,is leucine and X₃ is threonine. In certain embodiments, X₂ is aspartate,glutamate, alanine, glutamine, lysine or methionine. In certainembodiments, e.g., where sortase B is utilized, the recognition sequenceoften comprises the amino acid sequence NPX₁TX₂, where X₁ is glutamineor lysine; X₂ is asparagine or glycine; N is asparagine; P is prolineand T is threonine. In some embodiments selection of X may be based atleast in part in order to confer desired properties on the compoundcontaining the recognition motif. In some embodiments, X is selected tomodify a property of the compound that contains the recognition motif,such as to increase or decrease solubility in a particular solvent. Insome embodiments, X is selected to be compatible with reactionconditions to be used in synthesizing a compound comprising therecognition motif, e.g., to be unreactive towards reactants used in thesynthesis.

The invention contemplates use of sortase recognition motifs from any ofthe experimentally verified or putative sortase substrates listed athttp://bamics3.cmbi.kun.nl/jos/sortase_substrates/help.html, thecontents of which are incorporated herein by reference, and/or in any ofthe above-mentioned references. In some embodiments the sortaserecognition motif is selected from: LPKTG, LPITG, LPDTA, SPKTG, LAETG,LAATG, LAHTG, LASTG, LAETG, LPLTG, LSRTG, LPETG, VPDTG, IPQTG, YPRRG,LPMTG, LPLTG, LAFTG, LPQTS, it being understood that in variousembodiments of the invention the 5^(th) residue is replaced, asdescribed elsewhere herein. For example, the sequence used may be LPXT,LAXT, LPXA, LGXT, IPXT, NPXT, NPQS, LPST, NSKT, NPQT, NAKT, LPIT, LAET,or NPQS. The invention comprises embodiments in which ‘X’ in any sortaserecognition motif disclosed herein or known in the art is any standardor non-standard amino acid. Each variation is disclosed. In someembodiments, X is selected from the 20 standard amino acids found mostcommonly in proteins found in living organisms. In some embodiments,e.g., where the recognition motif is LPXTG or LPXT, X is D, E, A, N, Q,K, or R. In some embodiments, X in a particular recognition motif isselected from those amino acids that occur naturally at position 3 in anaturally occurring sortase substrate. For example, in some embodimentsX is selected from K, E, N, Q, A in an LPXTG or LPXT motif where thesortase is a sortase A. In some embodiments X is selected from K, S, E,L, A, N in an LPXTG or LPXT motif and a class C sortase is used. In someembodiments the first position of a sortase recognition motif is anaromatic amino acid (e.g., F or W) or an amino acid with a relativelysmall side chain such as A, D, S, P, and G, and a sortase variantcapable of recognizing the resulting motif is used. For example, in someembodiments L in LPXT is replaced by A, D, S, P, G, F, or W.

In some embodiments, a recognition sequence further comprises one ormore additional amino acids, e.g., at the N or C terminus. For example,one or more amino acids (e.g., up to 5 amino acids) having the identityof amino acids found immediately N-terminal to, or C-terminal to, a 5amino acid recognition sequence in a naturally occurring sortasesubstrate may be incorporated. Such additional amino acids may providecontext that improves the recognition of the recognition motif.

In some embodiments any of the sortase recognition sequences may furthercomprise one or more additional glycines or alanines at the C-terminus.It will be appreciated that a C-terminal amino acid of a polypeptide,e.g., a C-terminal amino acid of a polypeptide comprising a transamidaserecognition sequence, may be amidated, i.e., a C-terminal amino acid mayhave a —CONH₂ group instead of a —COOH group at the C-terminus incertain embodiments.

The term “transamidase recognition sequence” may refer to a masked orunmasked transamidase recognition sequence. An unmasked transamidaserecognition sequence can be recognized by a transamidase. An unmaskedtransamidase recognition sequence may have been previously masked, e.g.,as described in WO2010087994. In some embodiments, a “maskedtransamidase recognition sequence” is a sequence that is not recognizedby a transamidase but that can be readily modified (“unmasked”) suchthat the resulting sequence is recognized by a transamidase. Forexample, in some embodiments at least one amino acid of a maskedtransamidase recognition sequence has a side chain that comprises amoiety that inhibits, e.g., substantially prevents, recognition of thesequence by a transamidase of interest, wherein removal of the moietyallows the transamidase to recognize the sequence. Masking may, forexample, reduce recognition by at least 80%, 90%, 95%, or more (e.g., toundetectable levels) in certain embodiments. By way of example, incertain embodiments a threonine residue in a transamidase recognitionsequence such as LPXTG is phosphorylated, thereby rendering itrefractory to recognition and cleavage by SrtA. The masked recognitionsequence can be unmasked by treatment with a phosphatase, thus allowingit to be used in a SrtA-catalyzed transamidation reaction.

It will be appreciated that transamidase fragments having transamidationactivity can be utilized in the methods described herein. As describedin PCT/US2010/000274, such fragments can be identified by producingtransamidase fragments by known recombinant techniques or proteolytictechniques, for example, and determining the rate of protein or peptideligation. The fragment sometimes consists of about 80% of thefull-length transamidase amino acid sequence, and sometimes about 70%,about 60%, about 50%, about 40% or about 30% of the full-lengthtransamidase amino acid sequence such as that of S. aureus Sortase A(GenBank Accession number AAD48437). In some embodiments, the fragmentlacks an N-terminal portion of the full-length sequence, e.g., thefragment lacks the N-terminal portion extending to the end of themembrane anchor sequence (up to about amino acid 26). In someembodiments the fragment comprises the C-terminus of a full-lengthtransamidase amino acid sequence. In some embodiments, a catalytic coreregion from a sortase is utilized, e.g., a region is from about position60 to about position 206 of SrtA, e.g., S. aureus SrtA, or about fromposition 82 to about position 249 of SrtAstrep. Thus a sortase maycomprise or consist of a catalytic domain of a full length sortasepolypeptide. It will be appreciated that the polypeptide may alsocomprise an N-terminal methionine residue.

Transamidases from other organisms also can be utilized in the processesdescribed herein. Such transamidases often are encoded by nucleotidesequences substantially identical or similar to the nucleotide sequencesthat encode Srt A and Srt B. A similar or substantially identicalnucleotide sequence may include modifications to the native sequence,such as substitutions, deletions, or insertions of one or morenucleotides. Included are nucleotide sequences that sometimes are 55%,60%, 65%, 70%, 75%, 80%, or 85% or more identical to a native nucleotidesequence, and often are 90% or 95% or more identical to the nativenucleotide sequence (each identity percentage can include a 1%, 2%, 3%or 4% variance). One test for determining whether two nucleic acids aresubstantially identical is to determine the percentage of identicalnucleotide sequences shared between the nucleic acids.

Calculations of sequence identity can be performed as follows. Sequencesare aligned for optimal comparison purposes and gaps can be introducedin one or both of a first and a second nucleic acid sequence for optimalalignment. Also, non-homologous sequences can be disregarded forcomparison purposes. The length of a reference sequence aligned forcomparison purposes sometimes is 30% or more, 40% or more, 50% or more,often 60% or more, and more often 70%, 80%, 90%, 100% of the length ofthe reference sequence. The nucleotides at corresponding nucleotidepositions then are compared among the two sequences. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, the nucleotides aredeemed to be identical at that position. The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps, and thelength of each gap, introduced for optimal alignment of the twosequences.

Comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm.Percent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers & Miller, CABIOS 4: 11 17 (1989), whichhas been incorporated into the ALIGN program (version 2.0), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. Percent identity between two nucleotide sequences can bedetermined using the GAP program in the GCG software package (availableat www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set ofparameters often used is a Blossum 62 scoring matrix with a gap openpenalty of 12, a gap extend penalty of 4, and a frame shift gap penaltyof 5.

III. Methods of Sortagging and Processing Sortagged Cells

“Sortagging process” refers to a process in which at least some entities(e.g., proteins, cells) become sortagged. In general, a sortaggingprocess comprises contacting an entity to be sortagged, e.g., aeukaryotic cell, e.g., a mammalian cell, with a transamidase and asortase substrate under conditions in which a sortase-catalyzed reactioncan occur. In certain embodiments a sortase reaction is performed underphysiological conditions. In general sortase-catalyzed conjugation maybe performed by contacting a transamidase, acyl donor (sortasesubstrate), and nucleophilic acyl acceptor with one another undersuitable conditions to effect conjugation of the acyl donor to the acylacceptor. In embodiments of the present disclosure the nucleophilic acylacceptor may be a protein expressed by a mammalian cell. Contacting thecomponents with one another can be accomplished by adding them to onebody of fluid and/or in one reaction vessel, for example, or otherwiseplacing the components in close proximity to one another and allowingthem to collide. The components in the system may be mixed in a varietyof manners, such as by oscillating a vessel, if desired. The componentsmay be added in any order to the system. Conjugation may be performed inany convenient vessel (e.g., tubes such as microfuge tubes, flask,dish), microtiter plates (e.g., 96-well or 384-well plates), etc. Thereaction mixture may be maintained at any convenient temperature atwhich the reaction can be performed. In some embodiments, theconjugation is performed at a temperature ranging from about 3 or 4degrees C. to about 15 degrees C. In some embodiments, the conjugationis performed at a temperature ranging from about 15 degrees C. to about50 degrees C. In some embodiments, the ligation is performed at atemperature ranging from about 23 degrees C. to about 37 degrees C. Incertain embodiments, the temperature is room temperature (e.g., about 25degrees C.). Any convenient volume and component ratio may be utilizedto conjugate a sortase substrate to animal cells. In some embodiments,the acyl donor is present at a concentration ranging from about 5 μM toabout 10 mM, about 10 μM to about 5 mM, about 100 μM to about 500 μM,about 200 μM to about 1 mM. In some embodiments the concentration ofacyl donor is at least 0.25 mM, at least 0.5 mM, or at least 1 mM. Incertain embodiments, the transamidase is present at a concentrationranging from about 1 μM to about 500 μM, about 15 μM to about 150 μM,about 150 μM to about 250 μM, about 250 μM to about 500 μM. In certainembodiments, the transamidase is present at a concentration greater than10 μM, e.g., 11 μM to 20 μM, 20 μM to 30 μM, 30 μM to 50 μM, 50 μM to100 μM. In some embodiments a transamidase is present at a concentrationgreater than 10 μM and is a wild type sortase, e.g., a wild type sortaseA, e.g., a wild type S. aureus SrtA, or a variant thereof that has anactivity between 0.5-fold and 5-fold that of wild type S. aureus SrtA.In certain embodiments, the transamidase is present at a concentrationof about 10 μM to 1 mM, about 1504 to about 1 mM, about 20 μM to about 1mM, about 25 μM to about 1 mM, 30 μM to about 1 mM, about 50 μM to about1 mM, about 100 μM to about 1 mM, or about 250 μM to about 1 mM. Incertain embodiments, these concentrations apply to wild type sortase Ain particular, e.g., a wild type S. aureus sortase A.

In certain embodiments conjugation is performed in a reaction mixturecomprising an aqueous medium. Water with an appropriate buffer and/orsalt content compatible with cell viability may be used. One of ordinaryskill in the art will be familiar with a variety of buffers that couldbe used in accordance with the present invention. In some embodiments,the aqueous medium comprises calcium ions. For example, the aqueousmedium may contain between about 1.0 mM and about 50 mM calcium ions,e.g., from about 2 mM to about 25 mM calcium ions, e.g., from about 5 mMto about 15 mM calcium ions, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mM calcium ions. In certain embodiments, the aqueous medium containsbetween about 1 mM and about 5 mM calcium ions. In other embodiments,the aqueous medium contains greater than 0.5 mM or greater than 1 mMcalcium ions. In certain embodiments, the aqueous medium does notcontain substances that bind to, or sequester calcium ions or containsonly trace amounts of such substances, which would have negligibleeffects on the concentration of free calcium ions. In certainembodiments, the aqueous medium does not contain substances thatprecipitate calcium ions. In some embodiments, the aqueous medium doesnot include phosphate ions. In some embodiments, the aqueous medium doesnot include carbonate ions. In some embodiments, the aqueous medium doesnot contain chelating agents. For example, in some embodiments theaqueous medium does not contain substances that chelate calcium ions,such as EDTA or EGTA or contains only trace amounts of such substances,which would have negligible effects on the concentration of free calciumions. In some embodiments, if the medium contains a substance (otherthan a sortase) that binds to calcium ions, the concentration of suchsubstance is not sufficient to result in a decrease in the catalyticactivity of the sortase of more than 5%, 10%, 15%, 20%, or 25%. In someembodiments, the aqueous medium is prepared without addition of calciumions. In some embodiments the concentration of calcium ions is belowabout 1.0 mM, e.g., below about 0.5 mM, 0.25 mM, 0.1 mM, 0.05 mM, orlower. In some embodiments, suitable ligation conditions comprise pH inthe range of 6 to 8.5, 6 to 8, 6 to 7.5, 6.5 to 8.5, 7 to 8.5, 7.5 to8.5, 7.0 to 8.5, 7.3 to 7.8. It will be understood that theafore-mentioned concentrations, ratios, and conditions are exemplary andnon-limiting. Higher or lower concentrations and/or different conditionsmay be used in various embodiments.

When sortase substrates are conjugated to mammalian cells, reactionconditions that are compatible with cell viability and, in someembodiments, suitable to maintain normal cell function, are used.Appropriate conditions within the range of conditions described abovemay be used. For example, the temperature is within a suitable range formammalian cells, e.g., typically not more than 39 or 40 degrees,although higher temperatures, e.g., up to 45 degrees, may be used incertain embodiments. In some embodiments a temperature between about 10and 25 degrees C. may be used. In some embodiments a temperature betweenabout 25 and 37 degrees C. may be used. In some embodiments a relativelylow temperature, e.g., between about 4 and 10 degrees, may be used toreduce cellular metabolism and/or internalization of cell surfaceproteins. In embodiments in which non-mammalian cells are sortagged,reaction conditions that are compatible with cell viability and, in someembodiments, suitable to maintain normal cell function, are used.Appropriate conditions within the range of conditions described abovemay be used.

In some embodiments cells are at a concentration of between about 10⁵cells/ml and about 10¹² cells/ml, e.g., between about 10⁶ cells/ml andabout 10¹¹ cells/ml. A gentle means of mixing may be used if desiredsuch as gentle rocking. In some embodiments mammalian cells aresortagged in a composition comprising culture medium suitable forculturing the mammalian cell(s). In some embodiments the culture mediumis free or essentially free of serum, plasma, and/or animal tissue ororgan extracts. In some embodiments the culture medium contains serum orplasma. In some embodiments serum or plasma, if present, is from thesubject from whom the cells originated or to whom the cells are to beadministered. In some embodiments the culture medium is chemicallydefined.

Parameters such as the concentration of sortase, concentration ofsortase substrate, number and/or concentration of cells, ratio ofsortase substrate and/or sortase to cells, aqueous medium, and length oftime of the reaction may be selected based on a variety of factors, suchas the activity of the particular sortase, the nature of the sortasesubstrate (e.g., how readily it serves as a substrate for sortase), thedegree of conjugation desired, etc. In some embodiments the sortasereaction may be permitted to proceed for at least 15 minutes. In someembodiments the sortase reaction may be permitted to proceed for morethan 15 minutes. In some embodiments the sortase reaction may bepermitted to proceed for between about 30 minutes and about 24 hours,e.g., about 1-2, 2-4, 4-8, 8-12, 12-16, or 16-24 hours. In certainembodiments samples may be removed from a reaction vessel and tested forconcentration of unconjugated sortase substrate or reaction byproductand/or level of agent conjugated to cells. The reaction may be permittedto proceed until a desired endopoint is reached. In certain embodimentsthe number of cells is up to about 10¹⁴ cells, e.g., about 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ cells, or anyintervening range, e.g., between about 10⁵ and about 10¹² cells, betweenabout 10⁶ and about 10¹¹ cells, between about 10⁷ and about 10¹⁰ cells.In certain embodiments sortase-mediated modification of mammalian cellsmay be performed in 1-2 hours, or less (e.g., between 15 and 30 minutes,between 30 and 60 minutes). In some embodiments primary cells areobtained from a subject or from a donor, modified in vitro usingsortase, and at least some of the modified cells are administered to thesubject on the same day as the cells were obtained, or the followingday. In some embodiments the complete procedure, from cell harvesting toadministration of modified cells, may take between 4-12 hours, 12-24hours, or 24-48 hours. In certain embodiments cells that have a poorsurvival rate or lose functional activity or alter their phenotype whenmaintained in culture may be sortagged and administered to a subjectbefore losing viability or functional activity or exhibiting alteredphenotype.

In some embodiments a composition comprising one or more livingmammalian cells, sortase, and, in some embodiments, further comprising asortase substrate, is characterized in that at least 80%, 85%, 90%, 95%,97%, 98%, 99%, or more of the cell(s) remain viable in the compositionfor at least between 1-2 hours, 2-4 hours, 4-8 hours, 8-12 hours, ormore, e.g., at least 12-24 hours. In certain embodiments livingmammalian cells modified using sortase exhibit at most a 1%, 2%, 3%, 5%,10%, 15%, 20%, or 25% reduction in viability as compared to a suitablecontrol. In some embodiments, living mammalian cells modified usingsortase retain substantial functional activity. For example, in someembodiments cells subjected to a sortagging process exhibit at most a1%, 2%, 3%, 5%, 10%, 15%, 20%, or 25% reduction in at least onefunctional activity as compared to a suitable control. In someembodiments, living mammalian cells modified using sortase gain a newfunctional activity or have an increased functional activity as comparedwith a suitable control. For example, in some embodiments cellssubjected to a sortagging process may exhibit an increase of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in at least onefunctional activity as compared to a suitable control. In someembodiments cells subjected to a sortagging process may exhibit at leasta 2-fold, 5-fold, 10-fold, 20-fold, or more increase in at least onefunctional activity as compared to a suitable control. In someembodiments a new or increased functional activity is conferred by or asa result of an agent conjugated to the cells. In some embodiments afunctional activity may be measured in vitro. In some embodiments afunctional activity or change in a functional activity may be measuredafter administration of cells to a subject. In some embodiments afunctional activity is binding activity, cytokine secretion, cytotoxicactivity, phagocytic activity, antigen presenting activity, orcostimulation activity. In some embodiments, living mammalian cellsmodified using sortase may exhibit minimal or no detectable non-specificalteration (e.g., oxidation, denaturation, degradation) to cell surfaceproteins (other than those modified by sortase-mediated conjugation) ascompared with a suitable control. In some embodiments a suitable controlis cells of the same cell type or subtype that have not been contactedwith sortase. In some embodiments a suitable control is cells of thesame cell type or subtype that have been contacted with sortase in theabsence of a sortase substrate. In some embodiments control cells mayoriginate from the same culture, cell line, or subject as the cells withwhich they are compared. In some embodiments control cells not contactedwith sortase have been maintained under standard culture conditions forthat cell type or subtype (without sortase). In some embodiments asuitable control refers to a value (e.g., for viability or functionalactivity) measured for the cells prior to contacting them with sortase.In some embodiments control cells not modified using sortase have beenincubated in a composition without sortase for about the same length oftime and under substantially identical conditions as thesortase-modified cells with which they are compared.

In some embodiments a sortase substrate comprising an agent that has afunctional activity is conjugated to living mammalian cells withsortase. In some embodiments the agent retains at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99% or all of its functional activity ascompared to that of an unconjugated agent. In some embodiments afunctional activity is a catalytic activity, an inhibitory activity, abinding activity, or a cytotoxic activity. In some embodiments afunctional activity is an ability to stimulate a cell to survive,proliferate, become activated, differentiate, migrate, produce orsecrete one or more substances, exhibit a phenotypic characteristic(e.g., expression of one or more genes, cell surface marker phenotype),or attack a target cell. In some embodiments a functional activity is anability to inhibit a cell from proliferating, becoming activated,differentiating, migrating, producing or secreting one or moresubstances, dying, altering a phenotypic characteristic (e.g.,expression of one or more genes, cell surface marker phentopye), orattacking a target cell. In some embodiments an agent confers an abilityto participate in a new cell-cell interaction. In some embodiments anagent conjugated to a mammalian cell exerts an autocrine effect. Forexample, the agent may bind to a cell surface receptor expressed by acell to which the agent is conjugated and exert an effect on the cell.In some embodiments an agent exerts a paracrine effect. For example, theagent may bind to a cell surface receptor expressed by a cell locatednear the cell to which the agent is conjugated and exert an effect onthe cell. Binding of an agent to a cell surface receptor may modulate asignaling pathway, cause the cell to survive, differentiate, divide,migrate, maintain or acquire a functional activity, etc.

In some embodiments mammalian cells are subjected to a sortaggingprocess, and at least some sortagged cells are then separated fromsortase, unconjugated sortase substrate, and/or reaction byproduct.Separation may be performed using a variety of different methods and maybe based at least in part on size, charge, affinity, hydrophobicity,hydrophilicity, and/or other properties. In some embodiments sortase isimmobilized by attaching it to a support before or after being contactedwith mammalian cells. Immobilization may comprise contacting sortase ora composition containing sortase with an affinity reagent, e.g., anantibody, that binds to sortase, wherein the affinity reagent isattached to a support. In some embodiments the sortase is tagged, andthe affinity reagent binds to the tag. In some embodiments the supportis in a column, and a composition comprising cells and sortase is passedthrough the column. Sortase is retained by the column whereas cells passthrough. Cells may pass through the column at a different rate tounconjugated agent, thereby achieving separation. In some embodimentsthe agent comprises a tag that is removed during sortagging as part of areaction byproduct. Unconjugated sortase substrate and/or reactionbyproduct can be removed by an affinity agent that binds to the tag. Insome embodiments sortase is immobilized before being contacted withmammalian cells. For example, sortase may comprise a tag, e.g., a 6×-Histag, which may be used to immobilize the sortase to a metal-ioncontaining resin or substrate. Mammalian cells are incubated in thepresence of the immobilized sortase and an agent to be conjugatedthereto. Cells can readily be separated from the immobilized sortase. Insome embodiments sortase is immobilized to magnetic particles. It willbe understood that magnetic particles may be magnetisable andparamagnetic, e.g., superparamagnetic, i.e., they may only magnetic in amagnetic field.

In some embodiments at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the mammalian cells ina population of cells subjected to a sortagging process becomeconjugated with an agent. In some embodiments a population of cells isprocessed after sortagging to produce a composition in which at least5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or more of the mammalian cells are conjugated with anagent. In some embodiments mammalian cells that have been subjected tosortagging may be separated into two, three, four, or more groups, e.g.,between 2 and 10 groups, during or after being contacted with sortase ormay be subjected to selection. In some embodiments separation orselection produces a population of cells that is enriched in cellshaving a property that is desired and/or that is at least partlydepleted of cells having a property that is not desired, as comparedwith a starting population prior to separation.

In some embodiments cells that have been subjected to a sortaggingprocess are separated into two or more groups based at least in part onthe level of a moiety that has been conjugated thereto by sortase. Insome embodiments at least some cells that have a moiety conjugatedthereto at a level detectably greater than a suitable control level areseparated from cells that do not. In some embodiments cells that exhibitat least a specified level of moiety at their surface may be separatedfrom cells that exhibit a lower level or completely lack the moiety attheir surface. Groups may be defined based on the level of moiety usingany suitable classification system. In some embodiments cells aredivided into groups that are considered to exhibit low, intermediate, orhigh levels. In some embodiments the 1%, 5%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of cells having the highest level ofmoiety conjugated thereto are separated from the rest of the cells in apopulation. A level may be an absolute amount, relative amount, surfacedensity, volume density, or other suitable parameter. Suitableseparation methods may be utilized so as to produce a composition inwhich a selected degree of conjugation is achieved. For example, in someembodiments a composition comprises cells that have on average aspecified level or range of levels of moiety at their surface. Aspecified percentage may be a minimum percentage, e.g., at least 10%, ora range, e.g., between 10% and 50%, between 50% and 90%, etc.

Any suitable method may be used to detect or measure the level of anagent conjugated to cells or to separate cells into two or morepopulations if desired. One of ordinary skill in the art will be able toselect an appropriate method taking into consideration factors such as,for example, one or more physical, chemical, or biological properties ofthe agent such as affinity (e.g., binding affinity), charge,fluorescence, color, magnetic properties, mass, enzymatic activity. Insome embodiments a method utilizes fluorescence, affinity, or both. Forexample, in some embodiments an agent to be conjugated to cellscomprises a fluorescent moiety. Cells having the fluorescent moietyconjugated thereto may be detected or measured using, e.g., flowcytometry or immunofluorescence microscopy, and the level of agent maybe measured, if desired. Cells may be separated using fluorescenceactivated cell sorting (FACS). In some embodiments an agent is capableof binding to or being bound by a reagent (e.g. an antibody) comprisinga fluorescent label, and cells are contacted with such a reagent underconditions suitable for binding to occur, optionally followed by washingto remove non-specifically associated reagent. Cells are then subjectedto fluorescence activated cell sorting. In some embodiments anaffinity-based method is used. For example, in some embodiments an agentto be conjugated to cells comprises a tag. The tag may be detected usinga suitable reagent.

IV. Cells and Cell Culture

In general, any type of cells may be sortagged as described herein orused as a source of cells to be sortagged. In some embodiments cellscomprise or consist of mammalian cells. In some embodiments mammaliancells are primate cells (human cells or non-human primate cells), rodentcells (e.g., mouse, rat, rabbit, hamster cells), canine, feline, bovine,porcine, or other mammalian cells. In some embodiments cells are aviancells. In some embodiments cells are invertebrate animal cells. In someembodiments cells are fungal (e.g., yeast or mold) or protozoal cells.

A cell may be a primary cell, non-immortalized cell, immortalized cell,normal cell, abnormal cell, tumor cell, non-tumor cell, etc., in variousembodiments. A cell may originate from a particular tissue or organ ofinterest or may be of a particular cell type. In some embodimentsprimary cells may be freshly isolated from a subject. In someembodiments, cells are maintained in culture and may be passaged orallowed to double once or more following their isolation from a subject(e.g., between 1-5, 5-10, 10-20, 20-50, 50-80 passages or populationdoublings times) prior to use in a method disclosed herein. In someembodiments, cells have been passaged or permitted to double no morethan 1, 2, 5, 10, 20, or 50 times following isolation from a subjectbefore use in a method described herein. Cells “obtained from a subject”may comprise originally isolated cells and/or descendants thereof thatarise during culture of the originally isolated cells. In someembodiments cells are obtained from any tissue or organ of interest. Insome embodiments cells are obtained from a fluid such as blood, sputum,lymph, mucus, saliva, urine, blood, or lymph, from bone marrow, orlymphoid tissue (e.g., lymph node, spleen). In some embodiments cellsare obtained from connective tissue, muscle tissue, adipose tissue,epithelial tissue. In some embodiments cells are obtained from a tumor,site of infection, site of inflammation or immune-mediated tissuedamage, or lymphoid tissue that receives lymph from such a site (e.g.,nearest draining lymph nodes).

In some embodiments a cell is a member of a cell line. In someembodiments, a cell line is capable of indefinite proliferation inculture (immortal; immortalized). An immortalized cell line has acquiredan essentially unlimited life span, i.e., the cell line appears to becapable of proliferating essentially indefinitely. For purposes hereof,a cell line that has undergone or is capable of undergoing at least 100population doublings in culture may be considered immortal. Numerouscell lines are known in the art and may be used in various methodsdescribed herein. Cell lines can be generated using methods known in theart or obtained, e.g., from depositories or cell banks such as theAmerican Type Culture Collection (ATCC), Coriell Cell Repositories,Deutsche Sammlung von Mikroorganismen and Zellkulturen (GermanCollection of Microorganisms and Cell Cultures; DSMZ), EuropeanCollection of Cell Cultures (ECACC), Japanese Collection of ResearchBioresources (JCRB), RIKEN, Cell Bank Australia, etc. The paper andonline catalogs of the afore-mentioned depositories and cell banks areincorporated herein by reference.

In some embodiments a cell line, cell population, or cell culture isderived from a single cell. In some embodiments, a cell line, cellpopulation, or cell culture is derived from multiple cells. In someembodiments, cells of a cell line, cell population, or cell culture aredescended from a cell or cells originating from a single sample (e.g., asample obtained from a tumor) or individual. If desired, cells may betested to confirm whether they are derived from an individual or aparticular cell line by any of a variety of methods known in the artsuch as DNA fingerprinting (e.g., short tandem repeat (STR) analysis),single nucleotide polymorphism (SNP) analysis (which may be performedusing, e.g., SNP arrays (e.g., SNP chips) or sequencing), isoenzymeanalysis, human lymphocyte antigen (HLA) typing, chromosomal analysis,karyotyping, morphology, etc.

An appropriate cell donor, cell source, or cell line may be selectedbased on a variety of factors, such as the intended use of the cells,number of cells desired, availability, etc. In some embodiments, cellsare obtained from an individual who is healthy or is reasonably presumedto be healthy at the time the cells are obtained. In some embodiments,cells are obtained from an individual who does not have or is reasonablypresumed not to have one or more particular diseases, e.g., cancer,infection, autoimmune disease, at the time the cells are obtained. Insome embodiments cells are obtained from an individual who does not havea history of having a particular disease. In some embodiments cells areobtained from an individual who has or has had a particular disease. Insome embodiments the disease is cancer, a disease caused by a pathogen,or an autoimmune disease. In some embodiments the subject exhibitsresistance to a disease, e.g., a disease caused by a pathogen. In someembodiments the subject is recovering or has recovered from a disease.In some embodiments the subject is in need of treatment of a disease,e.g., cancer, a disease caused by a pathogen, an autoimmune disease, ora disease for which a transplant is indicated. In some embodiments cellsare obtained from an individual who is histocompatible with a subject inneed of treatment of such a disease.

Cells used in a method described herein may have been procured directlyfrom a subject or procured indirectly, e.g., by receiving the cells (orancestors of the cells) through a chain of one or more personsoriginating with a person who procured the cells (or ancestors of thecells) directly from the subject, e.g., by performing a biopsy, blooddraw, surgery, or other procedure on the subject. In some embodiments atleast some of the originally isolated cells may undergo one or morerounds of cell division. In some embodiments cells are obtained from atissue biopsy such as an excisional biopsy, incisional biopsy, or corebiopsy; a fine needle aspiration biopsy; a brushing; or a lavage. Insome embodiments cells are obtained from surgical or cellular samplesfrom a subject (e.g., tissue or cellular material harvested for purposesof obtaining tissue or cells, excess or discarded tissue or cellularmaterial, etc.). A surgical sample may be obtained from an organ or partof an organ that has been removed from a subject, e.g., because it isdiseased or injured or enlarged. Methods of obtaining samples andisolating cells from samples are well known in the art. In someembodiments cells are obtained from a tissue sample. In some embodimentscells are isolated from a tissue sample, by dissociation, e.g.,mechanical or enzymatic dissociation and may be collected, e.g., bycentrifugation, and washed, if desired. Cells can be subjected to avariety of procedures to select or enrich for cells of a desired celltype or having desired properties. In some embodiments enrichment isperformed at least in part based on expression (which may be lack ofexpression) of one or more cell surface markers using, e.g., FACS oraffinity reagents. One can select for against cells that expressparticular markers. In some embodiments enrichment is performed at leastin part by exposing cells to an agent or combination of agents (e.g.,cytokines) that promote differentiation and/or expansion of one or morecell types. In some embodiments a composition comprises at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more cells of a particulartype and/or expressing a particular marker or combination of markers. Insome embodiments at least some cells obtained from a subject and/ordescendants of such cells are stored, e.g., cryopreserved. Aliquots maysubsequently be thawed and used in one or more methods or compositionsdescribed herein. In some embodiments cells may be expanded in vitroprior to storage, after storage, or both.

In some embodiments cells may originate from any of the three germlayers: ectoderm, mesoderm, and endoderm. In some embodiments cells mayoriginate from any biological tissue of the four general classes ofbiological tissues: epithelial, muscular, connective, and nervoustissues. In some embodiments cells comprise epithelial cells.“Epithelium” refers to layers of cells that line the cavities andsurfaces of numerous structures in the body and is the type of tissuefrom which many glands are at least partly formed. Epithelial tissuesinclude, for example, tissues found in the gastrointestinal tract (e.g.,esophagus, stomach, small intestine, colon, rectum), liver, biliarytract, pancreas, respiratory tract (e.g., nasal passages, pharynx,larynx, trachea, bronchioles, bronchi, lungs, alveoli), oral cavity,skin, kidneys, ovaries, breast, prostate, cervix, uterus, bladder,ureter, testes, exocrine glands, endocrine glands, eye (e.g., retinalpigment epithelium, corneal epithelium, conjunctiva), blood vessels(vascular endothelim), lymph vessels (lymphatic endothelium), etc. Insome embodiments cells comprise or consist of muscle cells, e.g.,skeletal, smooth, or cardiac myocytes or myoblasts. In some embodimentscells comprise or consist of connective tissue cells, e.g., fibroblasts,adipocytes, cartilage cells (e.g., chondrocytes, chondroblasts), bonecells (e.g., osteoblasts, osteoclasts). In some embodiments cellscomprise or consist of nervous system cells, e.g., neural cells (e.g.,neurons), glial cells (e.g., astrocytes, oligodendrocytes, Schwanncells). In certain embodiments cells comprise or consist of pancreaticbeta cells, hepatocytes, keratinocytes, or melanocytes.

In some embodiments cells comprise hematopoietic cells. Hematopoieticcells include hematopoietic stem cells (HSCs), the blood cells that giverise to all other blood cells, and cells of the myeloid (e.g., monocytesand macrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells) and lymphoid (T cells, Bcells, NK cells) lineages. In some embodiments hematopoietic cells areobtained from peripheral blood, e.g., by venipuncture. In someembodiments hematopoietic cells are obtained by apheresis, a techniquein which the blood of an individual is passed through an apparatus thatseparates one or more constituents and returns the remainder to thecirculation. For example, erythrocytapheresis may be used to obtain redblood cells, leukapheresis may be used to obtain leukocytes (white bloodcells), plateletpheresis (also called thrombapheresis,thrombocytapheresis) may be used to obtain platelets. Separation mayemploy, e.g., centrifugation, elutriation, adsorbtion onto resin (e.g.,beads) with appropriate affinity, filtration, etc. In some embodimentscells are obtained peripheral blood after mobilization of such cells ortheir precursors, e.g., from bone marrow. For example, in someembodiments bone marrow hematopoietic stem cells (HSCs) are mobilizedby, e.g., injections of granulocyte-colony stimulating factor (G-CSF).In some embodiments cells, e.g., HSCs or other hematopoietic cells, areisolated from blood, e.g., peripheral blood or umbilical cord blood. Insome embodiments hematopoietic cells, e.g., HCSc, are isolated frommobilized peripheral blood. HSCs may be expanded ex vivo and/or may bedifferentiated ex vivo to yield lymphoid and/or myeloid cells of one ormore types.

In some embodiments cells comprise erythrocytes (red blood cells) and/orcommitted progenitors thereof. Red blood cells (RBCs) are typicallyabundant and readily accessible. In certain embodiments RBCs may be ofparticular interest as vehicles for delivery of therapeutic agents. Insome embodiments, for example, RBCs are sortagged with a therapeuticagent, e.g., a protein, a chemotherapy drug, an anti-infective agent,etc., and administered into the vascular system, e.g., intravenously.

In some embodiments cells comprise immune system cells. In someembodiments an immune system cell is a lymphocyte, monocyte, dendriticcell, macrophage, neutrophil, mast cell, eosinophil, basophil, naturalkiller (NK) cell, or mast cell. In some embodiments a lymphocyte is acell of the B cell lineage or T cell lineage. In some embodiments a Blymphocyte has rearranged its heavy (H) chain gene. In some embodimentsa B lymphocyte expresses a membrane-bound antibody. In some embodimentsa T cell expresses an alpha beta (αβ) T cell receptor (TCR). In someembodiments a T cell expresses a gamma delta (γδ) TCR. In someembodiments a T cell is a member of a T cell subset, e.g., a cytotoxic Tcell (also called killer T cell) or a helper T cell. Cytotoxic T cellsare typically positive for the cell surface marker CD8. Helper T cellsare typically positive for the cell surface marker CD4. In someembodiments a cell is a CD4+CD8-T cell. In some embodiments a cytotoxiccell is a CD4+ T cell, e.g., a CD4+CD8-T cell. In some embodiments acell is a CD4-CD8+ T cell. In some embodiments a cell is a CD4-CD8-Tcell. In some embodiments a cell is a CD4+CD8+ T cell. In someembodiments a T cell is a natural killer T (NKT) cell, e.g., aninvariant NKT (iNKT) cell. Natural killer T (NKT) cells are a subset ofT cells that display markers characteristic of both natural killer (NK)cells and T cells. NKT cells recognize lipid or lipid-containingantigens (e.g., glycolipids, lipopeptides) in the context of CD1molecules. NKT cells express an invariant TCRα chain rearrangement:Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβchains of limited diversity, and are sometimes referred to as canonicalor invariant NKT (iNKT) cells. Similar to conventional T cells, NKTcells develop from CD4-CD8-thymic precursor T cells following theappropriate signaling by CD1d. Human NKT cells can be stimulated andexpanded ex vivo by contacting them with α-galactosylceramide (α-GalCer)and a variety of cytokines.

In some embodiments a T cell is a regulatory T cell (Treg), e.g., aFoxP3+ regulatory T cell. In some embodiments a regulatory T cell is atype 1 regulatory (Tr1) cell, which does not express FoxP3. Tr1 cellstypically secrete interleukin 10 (IL-10) and transforming growthfactor-β (TGF-β), e.g., in response to antigenic stimulation. Tr1 cellsare capable of dampening autoimmunity and tissue inflammation partlythrough their secretion of IL-10. In some embodiments a T cell is afollicular helper T cell (T_(FH)). T_(FH) are antigen-experienced CD4+ Tcells found in the B cell follicles of secondary lymphoid organs such aslymph nodes, spleens and Peyer's patches and may be identified by theirconstitutive expression of the B cell follicle homing receptor CXCR5. Insome embodiments a T cell expresses and, in some embodiments secretes,one or more cytokine(s) and/or has a characteristic cell surface markerexpression profile. For example, in some embodiments a T cell has a Thelper 1 (Th1), T helper 2 (Th2), or T helper 17 (Th17) cytokinesecretion profile and/or cell surface marker profile. Th1 cells aretypically characterized by production of interferon-γ and TGF-beta. Th2cells may characteristically produce IL-4, IL-5, IL-6, IL-10, and IL-13.Th17 cells are typically characterized by production of interleukin-17.

In some embodiments a lymphocyte is a naïve cell (i.e., a cell that hasnot encountered an antigen to which its B cell receptor (BCR) or TCRbinds and is not descended from a lymphocyte that has encountered anantigen to which its BCR or TCR binds). In some embodiments an immunesystem cell has encountered, in culture or in vivo, an antigen to whichits BCR or TCR binds, or is descended from such a cell. In someembodiments an immune system cell has been activated, in culture or invivo. In some embodiments an immune system cell is activated by exposureto an antigen presenting cell (APC) that displays an antigen to whichthe cell's TCR or BCR binds and/or by exposure to one or more cytokines.In some embodiments an immune system cell having characteristics of anyof the afore-mentioned cell types may be generated at least in part invitro, e.g., by differentiation from a less differentiated or naïvecell. Protocols and reagents useful for generating such cells are knownin the art. Further information on varous immune cell types may be foundin, e.g., Zhu, J., et al., Differentiation of effector CD4 T cellpopulations. Annu. Rev. Immunol., 28 (2010), pp. 445-489; S. Crotty,Follicular helper CD4 T cells (TFH), Annu. Rev. Immunol., 29 (2011), pp.621-663.

In some embodiments a cell is a lymphokine-activated killer cell (LAK).LAKs are a heterogeneous population of cells consisting primarily of NK,NKT and T cells, which are generated in vitro by culture of peripheralblood mononuclear cells (PBMCs) in IL-2 (see, e.g., West, E J, et al.,British Journal of Cancer (2011) 105, 787-795 and references therein,e.g., Grimm E A, et al. J Exp Med 155(6): 1823-1841). The predominanteffector cells within LAKs are believed to be NK cells, but LAKs may bemore cytotoxic against tumour cells, including otherwise NK-resistanttargets, than typical peripheral blood NK cells (Grimm et al, 1982). Insome embodiments LAKs or other immune system cells may be sortagged withan agent comprising IL-2 or another cytokine or growth factor that mayexert an autocrine effect on the cells. Sortagging the cells may providean alternative to separately administering the cytokine or growth factor(e.g., systemically), which may reduce unwanted side effects that mightotherwise be associated with such administration.

In some embodiments a cell is an antigen presenting cell (APC). Anantigen-presenting cell (APC) is a cell that can process and displayforeign antigens in association with major histocompatibility complex(MHC) molecules on its surface. T-cells may recognize these complexesusing their T-cell receptors (TCRs). APCs may also display othermolecules (costimulatory proteins) that are required for activatingnaïve T cells. In some embodiments APCs express MHC class II molecules.Such APCs include dendritic cells, macrophages, and B cells. In someembodiments APCs are capable of stimulating CD4+ and CD8+ T cells. Insome embodiments APCs comprise professional APCs. In some embodimentsprofessional APCs are dendritic cells or macrophages. Dendritic cells(DCs) are a class of white blood cells that occur in most tissues of thebody, particularly those in contact with the exterior such as the skin(which contains a specialized dendritic cell type termed a Langerhanscell) and mucosal surfaces, as well as in the blood. During certaindevelopmental stages DCs grow membranous projections known as dendrites,from which the cell type gets its name. DCs serve as a link betweenperipheral tissues and lymphoid organs and play important roles inmodulating the activity of other immune system cells. Immature DCssample the surrounding environment for pathogens such as viruses andbacteria through pattern recognition receptors (PRRs) such as toll-likereceptors (TLRs). In response to stimuli such as pathogen components orother danger signals, inflammatory cytokines, and/or antigen-activated Tcells, they undergo maturation and migrate to the T cell area of lymphnodes or spleen, where they display fragments of previously phagocytosedand processed antigens at their cell surface using MHCII complexes. Aspart of the maturation process, DCs upregulate cell-surface receptorsthat act as co-receptors in T cell activation, such as CD80 (B7-1), CD86(B7-2), and/or CD40. DCs activate helper T cells (Th cells) bypresenting them with antigens derived from the pathogen in the contextof MHCII complexes, together with non-antigen specific costimulators.Binding of CD4+ expressed at the surface of Th cells to anon-polymorphic region of MHCII enhances the physical interactionbetween DC and Th cells, allowing potent stimulation of helper T cellsthat express TCR molecules capable of binding the peptide. In addition,DCs have the capacity to directly activate cytotoxic T cells and B-cellsthrough presentation of MHCII-peptide complexes and costimulators andare also able to activate the innate arm of anti-tumor immunity, e.g.,NK and NKT effector cells. DC stimulation promotes Th cellproliferation, activation, and differentiation into effector Th cells,memory Th cells, and regulatory Th cells. Effector Th cells provide“help” to cytotoxic T cells, B cells, and macrophages by, e.g.,secreting cytokines that exert a variety of stimulatory effects on thesecell types. Th help promotes proliferation and activation of cytotoxic Tcells, stimulates B-cell proliferation, induces B-cell antibody classswitching, and stimulates antibody production. Th stimulation alsoenhances the killing ability of macrophages. Memory T cells play animportant role in promoting the rapid mounting of a specific, strongadaptive immune response upon encountering an antigen to which a subjecthas previously been exposed. Regulatory Th cells are believed to play animportant role in the self-limiting nature of the immune response. Insome embodiments, DCs capable of presenting a particular peptidestimulate both the cell-mediated and humoral branches of the adaptiveimmune response towards targets containing that peptide as well asenhancing activity of the innate immune system. In some embodiments DCscomprise immature DCs, which lack one or more characteristics found inmature DCs present in tissues. For example, immature DCs may lackdendrites and/or lack one or more markers of mature DCs. In someembodiments immature DCs, e.g., immature human DCs, express and/or lackexpression of CD83. In some embodiments DCs, e.g., human DCs, comprisemyeloid DCs. In some embodiments DCs, e.g., human DCs, compriseplasmacytoid DCs. In some embodiments DCs comprise plasmacytoid CD303+DCs, myeloid CD1c+ DCs, and/or myeloid CD141+ DCs. In some embodimentsDCs, e.g., immature DCs, are obtained from the blood or generated invitro from peripheral blood mononuclear cells (PBMCs). See, e.g.,Tuyaerts, S., Cancer Immunol Immunother (2007) 56:1513-1537, fordiscussion of DC generation, antigen loading methods andimmunomonitoring approaches that may be used.

In some embodiments immune system cells are generated or expanded invitro from, e.g., HSCs or myeloid lineage progenitor cells.

In some embodiments a population of cells comprises immune system cellsof two or more types or subtypes, e.g., lymphocytes and DCs, CD4+ Tcells and CD8+ T cells, lymphocytes and NK cells, etc. Any combinationis encompassed. Two or more populations may be individually isolated andsubsequently combined. One or more of the populations, or the combinedpopulation, may be sortagged.

In some embodiments cells comprise peripheral blood mononuclear cells(PBMCs). As known in the art, PBMCs are peripheral blood cells that havea round nucleus, such as lymphocytes, monocytes, and NK cells. In someembodiments PBMCs are sortagged as a mixed population comprising two ormore distinct cell types or subtypes distinguishable by size,morphology, cell surface markers, and/or functional characteristics. Insome embodiments PBMCs are separated from other cells in a blood sample,so that at at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more ofthe cells are PBMCs. In some embodiments PBMCs are sortagged withoutfirst separating the PBMCs into two or more types distinguishable bysize, morphology, cell surface markers, and/or functionalcharacteristics. In some embodiments PBMCs may be separated into two ormore distinct populations, wherein one or more of the populations isenriched for one or more types of PBMC or is depleted of one or morePBMC types. In some embodiments the PBMCs are obtained from a subject towhom at least some of the PBMCs or their descendants are to beadministered after such cells are sortagged ex vivo. PBMCs can beisolated using standard methods known in the art, such as using Ficolldensity gradient centrifugation, which separates blood into a top layercontaining plasma and platelets, followed by a layer containing PBMCs,and a bottom fraction containing polymorphonuclear cells (such asneutrophils and eosinophils) and erythrocytes. The PBMC layer can beremoved, e.g., using a pipette, and sortagged. In some embodiments apreparation containing PBMCs isolated from blood may be further purifiedto remove residual red blood cells, e.g., prior to sortagging or aftersortagging. Red blood cell depletion from blood or from a preparationcontaining PBMCs isolated from blood may be performed by a variety ofmethods known in the art, such as osmotic shock, filtration, densitygradients such as ficoll-hypaque, percoll and hydroxyethyl starch andimmunoaffinity with monoclonal antibodies such as CD34 coupled tomagnetic beads. In some embodiments, T cells may be isolated from PBMC.In some embodiments a T cell subtype such as CD4+ or CD8+ T cells areisolated from PBMC. In some embodiments, NK cells may be isolated fromPBMC. In some embodiments PBMCs are depleted of one or more of cell suchtypes. In some embodiments, memory T cells, e.g., central memory Tcells, are isolated from PBMC.

In some embodiments a cell is an artificial APC (aAPC). Cellular aAPCmay be derived, e.g., from primary or transformed human or xenogeneiccells, e.g., fibroblasts or leukemia cells. In some embodimentsnon-mammalian cells, such as insect (e.g., D. melanogaster) cells may beused. Such cells may be engineered, e.g., using retroviral or lentiviraltransduction or other approaches such as transposon systems, to causethem to express molecules that provide TCR interaction, costimulatory,and adhesion events involved in immune synapse formation, allowing themto behave like naturally occurring APCs. Certain aAPCs are reviewed inKim, et al., Nature Biotechnology, 22(4): 403-410. In some embodiments,such aAPCs maybe used or sortagged in accordance with embodiments of thepresent invention. In some embodiments cells are engineered to coexpressany one or more of the following: a low affinity Fc receptor (e.g.,CD32), a high affinity Fc receptor (e.g., CD64), CD40, CD40L, CD70,CD80, CD83, CD86 (B7-2), ICOSL, GITRL, CD137L (4-1BBL), CD252 (OX40L),B7-H3, ICAM-1, LFA-3, and/or CD1. Some examples of geneticallyengineered aAPCs and methods of making and using them are described inMaus, M. V. et al. Ex vivo expansion of polyclonal and antigen-specificcytotoxic T lymphocytes by artificial APCs expressing ligands for theT-cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20, 143-148 (2002);Thomas, A. K., et al. A cell-based artificial antigen-presenting cellcoated with anti-CD3 and CD28 antibodies enables rapid expansion andlong-term growth of CD4 T lymphocytes. Clin. Immunol. 105, 259-272(2002). In some embodiments, cells may naturally express one or more ofthe afore-mentioned molecules and, optionally, are geneticallyengineered to express one or more additional afore-mentioned molecules.In some embodiments cells express at least two or all of the following:CD64, B7-2 (CD86), and CD137 ligand (CD137L). In some embodiments cellsexpress one or more cytokines, e.g., IL-2, IL-12, or IL-15. In someembodiments, cells express one or more membrane-bound cytokines, e.g.,membrane-bound IL-15.

In some aspects, the present disclosure contemplates use of sortaggingto modify an aAPC or cell to be used as an aAPC by conjugating any of avariety of agents to the cell surface. In some embodiments, cells aresortagged with (CD137L), membrane-bound IL-15, and/or other protein(s)that are normally expressed on the cell surface and may act as ligandsor interaction partners for receptors or other molecules expressed onother cells, e.g., cells to which an antigen is to be presented.Sortagging may be used in some embodiments to attach one or moreproteins to the cell surface instead of or in addition to causing thecells to express such proteins through use of genetic engineering.Sortagging may be used in some embodiments to attach moieties thatcannot be genetically encoded, such as lipids or small molecules,optionally complexed or attached to MHC proteins, CD1, or other proteinsthat normally present antigens. Such moieties may be presented asantigens or used for other purposes such as detection in vitro or invivo.

In some embodiments, cells to be used as aAPC are sortagged with anantigen of interest, resulting in aAPC that have an antigen of interestattached to their cell surface. In some embodiments, cells to be used asaAPC are genetically engineered to express an antigen of interest attheir cell surface, resulting in aAPC that have an antigen of interestexposed at least in part on their surface. The antigen of interest maybe modified to include a secretion signal sequence and transmembranedomain to cause it to be expressed as a cell surface protein. In someembodiments, the cells to be used as aAPC do not express HLA class Iand/or HLA class II molecules on their cell surface or at least do notexpress HLA-A, HLA-B, HLA-DQ, and HLA-DR. In some aspects, a lack ofsuch major histocompatibility (MHC) antigens prevents immune responsesthat may otherwise be directed against an aAPC that expresses such MHCantigens if the aAPC is cultured with non-allogeneic immune cells and/orintroduced into a non-allogeneic subject. In some embodiments cells tobe used as aAPCs are engineered to express an HLA class I molecule, HLAclass II molecule, or both. In some embodiments the cells do nototherwise express HLA class I, HLA class II, or both. In someembodiments, cells to be used as aAPC are contacted with a solubleantigen, e.g., in vitro. In some embodiments, the cells take up,process, and display the antigen or fragments thereof (e.g., peptides)on their surface in association with an HLA class I and/or class IImolecule. In some embodiments, cells to be used as aAPC are sortaggedwith an antigen presenting molecule (APM). For example, cells may besortagged with a molecule comprising at least a portion of an MHCprotein that is capable of binding to an antigen. In some embodimentsthe antigen presenting molecule has been contacted or is contacted(e.g., in vitro) with a peptide or other antigen such that the peptideor other antigen is bound to the APM. In some embodiments an APMcomprises at least a portion of an MHC protein that is capable ofbinding to an antigen, e.g., an MHC multimer or an HLA-Ig fusion protein(Oelke, 2003, full citation below).

aAPCs may be used for a variety of purposes, e.g., in the stimulation(activation and/or expansion) and/or positive and negative modulation ofcellular immune responses, e.g., of lymphocytes (T and/or B cells), NKcells, or other cells. aAPCs may be used in vitro, e.g., to activate andexpand immune cells that are normally stimulated by or require antigenpresentation (e.g., engagement of the TCR by antigen in an appropriatecontext, e.g., with provision of costimulatory signals by other cells)for maturation, proliferation, or acquisition of effector functions.aAPCs may be used, for example, for the in vitro stimulation of immunesystem cells that are to be administered to a subject. In someembodiments a composition comprises aAPC and immune cells to bestimulated. In some embodiments the composition further comprises atleast one cytokine (e.g., IL-2, IL-7, IL-12, IL-15, IL-21), anti-CD3monoclonal antibody such as OKT3, and/or anti-CD28 monoclonal antibody.In some embodiments the composition does not comprise anti-CD3 and/oranti-CD28 antibodies. In some embodiments, aAPCs are cultured in thepresence of anti-CD3 monoclonal antibody such as OKT3, and/or anti-CD28monoclonal antibody prior to contacting them with immune cells to bestimulated and are subsequently cultured in the presence of cells to bestimulated, optionally in the absence of anti-CD3 and/or anti-CD28antibodies. In some embodiments, the culture does not comprise furthercomprise feeder cells (other than the aAPCs and/or such cells as may beincidentally present in a population of cells to be stimulated by theaAPCs and may act as feeder cells, i.e., cells are not addedspecifically to act as feeder cells). In some embodiments, aAPC thatexpress an antigen to which a BCR, TCR, or CAR binds are used tostimulate the cells to cause them to proliferate and/or become activatedin vitro. In some embodiments aAPC are used in vivo. For example, insome embodiments aAPC are administered to a subject, e.g., to stimulateendogenous immune system cells or immune system cells administered tothe subject (or their descendants).

In some embodiments cells comprise phagocytic cells. Phagocytic cellsmay be professional or non-professional phagocytes. Professionalphagocytes include macrophages, monocytes, dendritic cells, mast cells,and neutrophils.

In some embodiments cells comprise adult stem cells. Adult stem cellsare undifferentiated cells that exist in post-natal, e.g., adult,organisms and are capable of giving rise to multiple different celltypes. Adult stem cells may be characterized by self-renewal (theability to go through numerous cycles of cell division giving rise todaughter cells at least one of which maintains an undifferentiatedstate) and multipotency or multidifferentiation potential, i.e., theability to give rise to descendants of multiple distinct cell types. Anadult stem cell may have multlineage potential and/or may be capable ofgenerating all the cell types of the organ or tissue from which itoriginates, potentially regenerating an entire organ from a few cells.Adult stem cells may undergo symmetric division, which gives rise to twoidentical daughter cells, both endowed with stem cell properties orasymmetric division, which produces one stem cell and a progenitor cellwith more limited self-renewal potential. Progenitor cells can gothrough at least one round of cell division, typically several rounds ofcell division, before differentiating into a mature cell. Examples ofadult stem cells include, e.g., hematopoietic stem cells, neural stemcells, endothelial stem cells, intestinal stem cells, mammary stemcells, mesenchymal stem cells, neural crest stem cells. Adult stem cellsmay be further differentiated using appropriate protocols known in theart. In some embodiments cells comprise or consist of gametes (egg orsperm cells) or germ cells. In some embodiments cells comprisepluripotent cells, e.g., embryonic stem cells. In some embodimentsmesenchymal stem cells (MSCs) comprise adherent non-hematopoietic bonemarrow-derived stem cells. In some embodiments MSC may be characterizedby: plastic adherence, maintenance of tri-lineage (osteogenic,adipocytic, and chondroblastic differentiation potential after in vitropropagation), and lack of the hematopoietic markers CD45, CD34, CD14,CD11b, CD79-a, CD19, and HLA-DR, and simultaneous expression of thesurface molecules CD73, CD90, and CD105 on at least 95% of thepopulation (Dominici M et al., (2006) Minimal criteria for definingmultipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy 8:315-317).

In some embodiments cells may be generated in vitro by reprogramming asomatic cell to a less differentiated state or by reprogramming asomatic cell from a first differentiated state to a seconddifferentiated state (sometimes termed “transdifferentiation”). In someembodiments reprogramming comprises reprogramming a cell to amultipotent or pluripotent state. In some embodiments a reprogrammedcell is an induced pluripotent stem (iPS) cell. In some embodiments aniPS cell may be generated by causing the cell to express or contain oneor more genetic factors (“reprogramming factors”). Suitable combinationsof reprogramming factors are known in the art. In some embodimentsreprogramming factors include one or more factor selected from Oct4,Sox2, Klf4, Nanog, Lin28, and c-Myc. Examples of suitable combinationsinclude, e.g., (1) Oct4, Klf4, Sox2, and optionally c-Myc; (2) Oct4,Sox2, Nanog and Lin28; (3) Oct4, Esrrb, Nanog; (4) Sox2, Sal14, Nanog;(5) Lin28, Sal14, Esrrb, Nanog; (6) Lin28, Sal14, Esrrb, Nanog. Nanogmay be replaced by Dppa2. A variety of small molecules that enhancereprogramming and/or substitute for or induce expression of one or morereprogramming factors are known and may be used in various embodiments.Such molecules may, e.g., activate or inhibit one or more signalingpathways or may cause alterations in chromatin structure. In someembodiments a somatic cell to be reprogrammed is a fibroblast,keratinocyte, or hematopoietic cell. In some embodiments a somatic cellis reprogrammed using Oct4 and at least one small molecule. In someembodiments nucleic acid(s) encoding one or more reprogramming factorsoperably linked to regulatory elements capable of directingtranscription is introduced into cells using a viral vector, e.g., aretroviral vector. A copy of the coding sequence(s) may integrate intothe genome. In some embodiments such nucleic acid is subsequently atleast partially excised from reprogrammed cells. In some embodimentsreprogramming is performed using a method that does not involve alteringthe genome of a cell. For example, translatable RNA encoding one or morereprogramming factors may be introduced into the cell and/or a cell maybe contacted with one or more small molecules that promotereprogramming. In some embodiments translatable RNA may be syntheticmodified RNA. Methods that may be used for reprogramming using syntheticmodified RNA are described, e.g., in PCT/US2011/032679 (WO2011130624)and/or in Mandal P K, Rossi D J. Reprogramming human fibroblasts topluripotency using modified mRNA. Nat Protoc. 2013; 8(3):568-82. In someembodiments, reprogramming mammalian somatic cells may comprisestressing the cells by, e.g., transient exposure to chemical or physicalstimuli such as low-pH conditions (e.g., about pH 5.7). The cells maythereby be stimulated to undergo stimulus-triggered acquisition ofpluripotency (STAP), and cell lines comprising pluripotent cells may bederived under appropriate conditions (see, e.g., Obokata, H., et al.,Stimulus-triggered fate conversion of somatic cells into pluripotency.Nature, 2014; 505 (7485): 641-647; and Obokata, H., et al.,Bidirectional developmental potential in reprogrammed cells withacquired pluripotency. Nature, 2014; 505 (7485): 676-680). In someembodiments lymphocytes are reprogrammed via STAP. In some embodimentsthe reprogramming does not entail introducing exogenous nucleic acids orproteins into the cells. In some embodiments a cell that has beenreprogrammed to a less differentiated state, e.g., to pluripotency isinduced to differentiate into one or more selected cell types or alongone or more selected cell lineages. For example, a pluripotent cell maybe induced to have properties of an adult stem cell. Such cells may befurther differentiated using appropriate protocols known in the art.Adult stem cells or cells differentiated therefrom may be sortagged asdescribed herein. In some embodiments, for example, pluripotent cellsgenerated from somatic cells may be reprogrammed to hematopoietic cells,e.g., immune system cells, e.g., T cells, B cells, NK cells.

Cells may be cultured in any suitable cell culture vessel or using anysuitable cell culture system. In some embodiments plates (e.g.,multiwell plates) or flasks, e.g., conventional tissue cultureplasticware, may be used. A cell culture system may comprise means forregulating oxygen and/or carbon dioxide concentration, pH, or other cellculture relevant parameters. In some embodiments a cell culture system,sometimes termed a cell bioreactor, that provides mechanical rocking orstirring or pumping (e.g., sparging) to perfuse media with gas or thatprovides continuous or intermittent media flow or exchange may be used.The use of such systems may enhance cell expansion, which may result inhigher cell densities than typically attained using conventionalplasticware. A variety of cell culture systems (e.g., hollow fiberbioreactors, stirred tank bioreactors, bags, etc.) useful for culturingcells are known in the art. In some embodiments a gas-permeable bag orvessel comprising a gas-permeable membrane, e.g., a silicone membrane,may be used. In some embodiments Vuelife™ bags (Cellgenix, Freiburg,Germany), a WAVE Bioreactor™ system (GE Health, Uppsala, Sweden),BIOSTAT® CultiBag RM system (Sartorius Stedim Biotech, Göttingen,Germany), or G-Rex system (Wilson Wolf Manufacturing, New Brighton,Minn.), or system employing similar technology, may be used. In someembodiments, for example, a cell culture system may comprise a cellculture vessel comprising a gas-permeable cultureware flask in which O₂and CO₂ are exchanged across a gas-permeable membrane at the base of theflask. In some embodiments a cell culture vessel may have a volume orrecommended media volume of, e.g., up to 5 ml, 10 ml, 15 ml, 20 ml, 25ml, 30 ml, 40 ml, 50 ml, 75 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml,750 ml, or 1,000 ml. In some embodiments a cell culture vessel may havea volume or recommended media volume of more 1 liter (1), e.g., up to1.5, 2.0, 2.5, 3.0, 4.5, 5.0 liters, or more. In some embodiments aselected number of cells may be cultured produced in a single culturevessel or system or in some embodiments using multiple culture vesselsor systems. In some embodiments a selected number of cells may becultured produced without need for stirring. In some embodiments aselected number of cells may be cultured produced without need formedium change, or with only 1, 2, 3 medium changes. In some embodimentsa selected number of cells may be, e.g., up to about 10¹⁴ cells, e.g.,about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴cells, or any intervening range, e.g., between about 10⁵ and about 10¹²cells, between about 10⁶ and about 10¹¹ cells, between about 10⁷ andabout 10¹⁰ cells. In some embodiments between about 10⁵-10⁸ cells andabout 10¹¹-10¹³ cells are cultured or produced. In some embodiments asystem allows for medium changes without removing a lid. In someembodiments cell culture is performed under aseptic conditions. In someembodiments cells may be cultured on microcarriers.

In some embodiments cells are obtained, e.g., from a subject, andexpanded in vitro. Cell “expansion” refers to an increase in the numberof cells. In some embodiments cells are cultured for between about 48hours and about 12 weeks, e.g., between 2 and 6 weeks. In someembodiments the number of cells is increased by a factor of at least 2,e.g., between 2 and 100, between 100 and 500, between 500 and 1,000,between 1,000 and 5,000, between 5,000 and 10,000, between 10,000 and50,000, between 50,000 and 100,000, or more, relative to the number ofcells in an initial sample or culture. In some embodiments expandedcells substantially retain or exhibit a particular phenotype, functionalactivity, or cell surface marker profile of interest. In someembodiments an expanded culture may be subjected to sorting orseparating or selection to enrich for cells having a selected phenotype,functional activity, or cell surface marker profile. In some embodimentsan expanded culture may be characterized for a selected phenotype,functional activity, or cell surface marker profile.

Protocols and useful reagents and culture systems suitable for culturingand/or expanding a wide variety of cell types are known in the art. Insome embodiments a cell culture medium, cell culture system, sortasepreparation, sortase substrate, cell culture process, or sortaggingprocess complies with Good Manufacturing Practices (GMP). In someembodiments a sortase preparation that is free or substantially free oressentially free of endotoxin may be used. In some aspects, the presentdisclosure provides GMP-compliant compositions comprising one or morecells that have an agent conjugated by sortase to a non-geneticallyengineered endogenous polypeptide expressed by the cell. In someembodiments cell culture, sortagging, or both, are performed underconditions appropriate to permit subsequent introduction of thesortagged cells into a human subject. In some embodiments a cellcomposition may be subjected to any one or more tests used in the art toassess suitability for administration to humans and/or for veterinarypurposes. In some embodiments a cell composition satisifies any one ormore criteria used in the art to assess suitability for administrationto humans and/or for veterinary purposes. In some embodiments cultureconditions include use of culture medium that is free or essentiallyfree of serum, plasma, and/or cell and tissue-derived substancesextracts. In some embodiments culture conditions may include absence offeeder cells. In some embodiments, if one or more such substance(s) orfeeder cells is used, they have been appropriately tested to confirmthat they are free of human pathogens and/or substances that wouldrender them potentially unsafe or unsuitable for administration to humansubjects. In some embodiments, if one or more such substance(s) orfeeder cells is used, it is obtained from the same subject as that fromwhich the cells are obtained or a subject to whom the cells are to beadministered. In some embodiments one or more recombinantly producedproteins may be used. In some embodiments the composition comprises achemically defined culture medium. Suitable culture medium may beobtained from a variety of commercial suppliers, e.g., StemcellTechnologies Inc. (Vancouver, BC, Canada), Life Technologies, Inc.(Carlsbad, Calif.), Lonza (Basel, Switzerland). For example, in someembodiments StemSpan medium may be used, e.g., to culture hematopoieticcells. In some embodiments AIM V® Medium may be used, e.g., to cultureimmune system cells such as lymphocytes, monocytes, dendritic cells,natural killer cells, PBMC, macrophages, etc. In some embodiments GIBCOOpTmizer™ CTS™ T-Cell Expansion Serum Free Medium (Life Technologies) isused, e.g., for culture of human T lymphocytes. In some embodimentsX-VIVO™ medium (Lonza), e.g., X-VIVO™ 10, X-VIVO™ 15, or X-VIVO™ 20 isused, e.g., to culture immune system cells such as lymphocytes,monocytes, dendritic cells, natural killer cells, PBMC, macrophages,etc. In some embodiments RPMI, DME, or DMEM may be used. One of ordinaryskill in the art will be aware of suitable culture media for cell typesof interest.

As used herein, a composition may be considered “free” of a particularmaterial or substance if the material or substance is not deliberatelyadded to or known to be present in the composition and/or isundetectable using standard methods used in the art for detection ofsuch material or substance and/or if the composition has been preparedunder conditions accepted in the art as sufficient to achieve absence ofthe material or substance. In some embodiments a composition may besubstantially free or essentially free of any one or more materials orsubstances. In some embodiments “essentially free” refers to aconcentration of no more than 0.1%. 0.05%, 0.01%, 0.005%, 0.001%,0.0005% of such material or substance by weight (e.g., dry weight),volume, or by moles. In some embodiments “substantially free” refers to1% or less, e.g., 0.5% or less, e.g., 0.2% or less of such material orsubstance by weight (e.g., dry weight), volume, or by moles. In someembodiments a composition is considered substantially free of a materialor substance, e.g., an adjuvant, if the component or substance is notdetectable using a standard detection method used in the art fordetecting such material or substance. In some embodiments a compositionis prepared without deliberately including a substance, e.g., anadjuvant. In some embodiments a composition is prepared withoutdeliberately including an adjuvant in an amount that would be effectiveto enhance an immune response when the composition is contacted withcells in vitro or in vivo.

In some embodiments cells cultured or sortagged in the compositionsatisfy regulatory requirements for administration to a human subject.In some embodiments cells cultured or sortagged in the compositionsatisfy regulatory requirements of a government agency such as the USFood and Drug Administration, European Medicines Evaluation Agency, or asimilar agency responsible for evaluating the safety of therapeuticagents prior to their administration to humans or being placed on themarket for administration to humans. In some embodiments cells arewashed or otherwise processed to remove media components aftersortagging, e.g., prior to administration to a subject. In someembodiments a composition is tested and determined to be free ofpathogens that may infect humans. In some embodiments a test may utilizePCR or a biological assay for presence of a particular pathogen.

Methods for culturing, expanding, and, in some embodiments, activating,cells of various types are known in the art. For example, T cells may beexpanded and activated using antibodies that bind to CD3 (e.g.,Muromonab-CD, also known as Orthoclone OKT3, “OKT3”), optionally incombination with one or more cytokines such as IL-2. In some embodimentsimmune system cells may be exposed to costimulatory signals provided bysoluble, surface-bound, or cell-bound (e.g., APC-bound) costimulatorymolecules, such as CD28, for example. In some embodiments immune systemcells may be contacted in vitro with an antigen or epitope, optionallyin association with an MHC protein. Hematopoietic stem cells may beculturing and/or expanded as described, e.g., in US Pat. Pub. Nos.20110117061; 20110136230; and/or 20110196343. Such cells may bedifferentiated along various hematopoietic lineages if desired.

Examples of systems and/or protocols that may in various embodiments beused to culture, and, in some embodiments, expand and/or activate, cellsare described in PCT/US2002/028161 (WO/2003/024989); PCT/US2004/001349(WO/2004/065590); PCT/US2008/062687 (WO/2009/136907); PCT/US2009/049944(WO/2010/006055); PCT/US2010/046505 (WO/2011/028531); PCT/NL2006/000319(WO/2007/001173); PCT/US2010/061706 (WO/2011/079165); PCT/EP2012/063034(WO/2013/007574); PCT/SE2010/050333 (WO/2010/110734); Digiusto, D L andCooper, L J N, Cytotherapy. 2007; 9(7):613-29; Sutlu, T., et al.,Cytotherapy. 2010 December; 12(8):1044-55; Spanholtz J, et al., PLoSOne. 2011; 6(6):e20740. doi: 10.1371/journal.pone.0020740, Levine, B. etal., J Hematother. (1998) 7(5):437-48; Tumaini, Cytotherapy. 2013;15(11):1406-15, and references in any of the foregoing).

In some embodiments cells are separated into two or more groups prior tosortagging, and only one or some (but not all) of the groups aresubjected to a sortagging process. For example, in some embodimentscells of a particular cell type may be selected for sortagging from astarting population comprising cells of multiple types. Cells of othertypes that may be present in the starting population may, for example,be used for other purposes, stored (e.g., cryopreserved), discarded, orsubsequently combined with sortagged cells. Cells may be selected basedon any one or more properties. A property may be any phenotypiccharacteristic of a cell, cell type, or cell state, e.g., anycharacteristic that may be observed or detected, or any combination ofsuch characteristics. Examples include, e.g., morphologiccharacteristics; physical, biochemical, or physiological properties;biological behavior, the presence, absence, or level of gene expressionproducts such as RNA or proteins. In some embodiments a phenotypiccharacteristic is quantifiable. In some embodiments cells may beseparated based on size, light scattering, density, binding affinity forone or more substances, expression of at least one gene as assessed,e.g., by the level of a gene product of the gene.

Any suitable separation method(s) may be used. Multiple steps ofselection or separation may be used. In some embodiments selectioncomprises at least one positive selection, wherein desired cells areretained or enriched for based on one or more properties of the cells ofinterest (wanted cells; desired cells), e.g., using a binding agent thatbinds to cells of interest. In some embodiments selection may compriseat least one negative selection, wherein cells that are not of interest(unwanted cells; undesired cells) are removed or depleted based on oneor more properties of the unwanted cells (e.g., using a binding agentthat binds to unwanted cells). Examples of useful separation methodsinclude centrifugation, elutriation, contacting with an affinity resin(e.g., beads) that retains or removes unwanted cells, flow cytometry,fluorescence activated cell sorting (e.g., after contacting the cellswith appropriately labeled reagents that bind to cell surface markerscharacteristic of cells whose removal or retention is desired).

In some embodiments cells having a particular cell surface markerexpression profile are selected or removed. A cell surface markerexpression profile may comprise presence, absence, and/or level of anyone or more cell surface markers. Cell surface marker expressionprofiles characteristic of many different cell types or cell subsetshaving various functional characteristics of interest are known in theart. In some embodiments a cell surface marker comprises a cluster ofdifferentation (CD) molecule. In some embodiments an antibody-basedenrichment procedure is used, which may be combined with columns,magnet-based separation, and/or centrifugation. In some embodimentsmagnetic particles (particles, whether or not magnetic, are sometimestermed “beads”) may be used. Examples of such particles include thoseknown as Dyabeads (Life Technologies, Carlsbad, Calif.), MACS microbeads(Miltenyi Biotech, Auburn, Calif.). In some embodiments, cells in asingle-cell suspension are magnetically labeled with beads that have anappropriate binding agent (e.g., an antibody) attached thereto. Thesample is applied to a column placed in a magnetic separator. Theunlabeled cells pass through while the magnetically labeled cells areretained within the column. The flow-through can be collected as theunlabeled cell fraction. The column may be washed and removed from theseparator, and the magnetically labeled cells eluted from the column.Thus both labeled and unlabeled cells can be isolated if desired. Insome embodiments beads may have any appropriate dimensions, e.g.,diameter, volume. In some embodiments desired cells are obtained withoutusing columns, without using magnets, and/or without labeling desiredcells with a binding reaget such as an antibody. For example, wholeblood may be contacted with antibody complexes that comprise two or moreantibodies to antigen(s) expressed at the surface of red blood cells butnot expressed at the surface of desired cells and one or more antibodiesto antigen(s) expressed at the surface of cells to be removed. In someembodiments a tetrameric antibody complex may be used. The antibodycomplexes crosslink undesired cells to red blood cells present in thewhole blood, resulting in complexes (sometimes termed immunorosettes)that can be removed by centrifugation, e.g., over an appropriate densitymedium (e.g., Ficoll™). Centrifugation may be used to pellet theimmunorosettes, thereby removing the unwanted cells along with red bloodcells and leaving desired cells. The purified cells are present as anenriched population at the interface between the plasma and the buoyantdensity medium. Reagents suitable for performing such separation arecommercially available. For example, RosetteSep® reagents or kits may beused (Stemcell Technologies) may be used. This technology utilizestetrameric antibody complexes that crosslink unwanted cells to multiplered blood cells already present in the sample, forming immunorosettes.When centrifuged over the appropriate density medium (e.g. Ficoll), theunwanted (rosetted) cells pellet along with the red blood cells, leavingthe desired cells untouched and highly enriched at the density medium:plasma interface. RosetteSep™ may be used on its own with standarddensity gradient centrifugation or with a specialized cell processingtube (SepMate™). Suitable RBC antigens include, e.g., glycophorin, e.g.,glycophorin A. In some embodiments isolation of desired cells from amixed sample is performed using a procedure that takes no more than 30minutes or no more than 60 minutes. In some embodiments separation maybe performed at least in part using an automated system. For example,RoboSep™ (Stemcell Technologies) is an instrument that provides forautomation of immunomagnetic cell separation performing the stepsnecessary to magnetically label and separate virtually cells of aselected cell type by positive or negative selection. In someembodiments a selection procedure may comprise culturing cells underselective conditions, wherein the selective conditions kill or inhibitproliferation of cells having certain characteristics that are notdesired. In some embodiments selective conditions comprise culturing acell population comprising multiple cell types in the absence of one ormore factors required for survival or proliferation of unwanted cells.

In some embodiments mammalian cells may be assessed forimmunocomptibility with a subject to whom they are to be administered.Cells may be deemed immunocompatible if they are unlikely to provoke asignificant immune response in a subject to whom they are administered,e.g., an immune response that would materially reduce the viabilityand/or functional activity of the cells or produce excessive symptoms ina subject. In some embodiments immunocompatiblity compriseshistocompatibility. Histocompatibility may be assessed using anysuitable method known in the art. In some embodiments histocompatibilitytesting comprises determining whether a potential recipient hasantibodies to HLA antigens expressed by cells to be administered and/orwhether a potential recipient has an HLA genotype that differs from thatof the cells to a sufficient extent as to be deemed incompatible (e.g.,likely to be or at risk of being subject to attack by the recipient'simmune system) as reasonably determined by one of ordinary skill in theart. In some embodiments cells to be administered to a human subject aretested to determine their HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/orHLA-DR genotype and/or a sample from a subject is tested to determinewhether it contains antibodies to MHC Class I and/or MHC Class IIantigens of cells to be administered to the subject. In some embodimentscells are deemed histocompatible for administration to a subject if theyhave at least the same HLA-A, HLA-B, and HLA-DR alleles as the subject'scells and/or if the subject's blood does not harbor antibodies to MHCmolecules expressed by the cells. Methods of testing forhistocompatibility include, e.g., screening for preformed alloreactiveantibodies using complement-dependent cytotoxicity assays (CDC) whichdetects complement-activating IgG1/3 and IgM antibodies using a panel ofHLA-typed lymphocytes to identify reactive antibodies, solid-phasemethods such as ELISA, and flow cytometry-based techniques (usingclassical flow cytometry or Luminex). For example, Luminex-basedantibody screening technology uses purified HLA antigens immobilized ona panel of microbeads. These HLA molecules on the microbeads are targetsfor HLA-specific antibodies in a given sample. Anti-HLA antibodies ofthe IgG isotypes which are bound to the microbeads are detected by asecondary IgG-specific antibody which may conjugated with a label suchas R-phycoerythrin (PE). Nucleic acid-based (e.g., DNA-based) tissuetyping may be performed using, e.g., sequence-specific primers and/orsequence-specific probes (see, e.g., Dunckley H. HLA typing by SSO andSSP methods. Methods Mol Biol. 2012; 882:9-25). Primers may be used toamplify relevant sequences, e.g., using PCR. Probes may be attached tooligonucleotide arrays or beads. In some embodiments sequencing, e.g.,high throughput sequencing, may be used for genotyping. In someembodiments techniques generating low-resolution (i.e. 2-digits HLAnomenclature) results or high-resolution (i.e. at least 4-digits HLAnomenclature) results may be used. Intermediate HLA resolution comprisesa 2-digits HLA nomenclature with supplemental characters to definegroups of HLA alleles. In general, an HLA typing resolution and/ordegree of HLA matching at a level relevant for different clinicalapplications may be used.

In some embodiments immunocompatible red blood cells are of the sameblood group as an individual to whom such cells are to be administered(e.g., at least with respect to the ABO blood type system and, in someembodiments, with respect to the D blood group system) or may be of acompatible blood group. For example, in some embodiments type O Rh Dnegative blood may be administered to an individual of any ABO bloodgroup (i.e., A, B, O or AB), and persons with type O RhD negative bloodmay be considered “universal donors”. Methods for determining bloodgroups are well known in the art. In some embodiments red blood cellsare cross-matched with a potential recipient of the cells.Cross-matching may comprise mixing a sample of the recipient's serum,plasma, or blood with a sample of the red blood cells to potentially beadministered and checking if the mixture agglutinates. If agglutinationis not observed, the RBCs may be considered to match the recipient.

In some embodiments a eukaryotic cell that comprises a sortagged,endogenous, non-genetically engineered polypeptide is a geneticallyengineered cell. In some embodiments the cell has not been geneticallyengineered for sortagging. In some embodiments the cell has beengenetically engineered before being sortagged. In general, the cell maybe any genetically engineered eukaryotic cell. The cell may be amammalian cell, fungal cell, insect cell, protozoal cell, or othereukaryotic cell. The cell may be of any cell type, e.g., any cell typedescribed herein. For example, in some embodiments the geneticallyengineered cell is an immune system cell, e.g., a T cell, B cell, NKcell, dendritic cell, monocyte, or macrophage. In general the cell maybe genetically engineered for any purpose, in any way, and using anymethod known in the art. A genetically engineered cell may comprisemultiple genetic alterations of any one or more type(s), e.g., one ormore insertions into genomic DNA, one or more deletions of genomic DNA,or both. A polypeptide or noncoding RNA encoded at least in part byexogenous DNA integrated into the genome of a cell or encoded by anendogenous gene whose sequence has been modified by genetic engineeringmay be referred to as a “recombinant gene product”.

In general, genetic engineering comprises introducing one or moreexogenous nucleic acids into a cell. Nucleic acids can be introducedinto cells using transfection (e.g., using any of a variety oftransfection reagents), electroporation, virus-mediated nucleic acidtransfer, etc. One of ordinary skill in the art will select appropriatemethods, vectors, expression control elements, etc., to achieve desiredalterations in the genome of a cell and/or to achieve expression ofdesired proteins and/or RNAs. For example, if a virus is used as avector, an appropriate method may comprise contacting a mammalian cellwith the virus under conditions appropriate for the virus to enter thecell. It will be understood that, depending on factors such as thevector and the particular method, a nucleic acid introduced into a cellmay be at least in part copied or reverse transcribed, and such copy ora portion thereof may be inserted into the genome. It will also beunderstood that use of the term “inserted” is not intended to imply orrequire any particular mechanism and encompasses processes mediated byretroviral integrase, homologous or non-homologous recombination,creation of breaks in genomic DNA that are repaired by endogenous DNArepair mechanisms, or any other process that results in addition of oneor more nucleotides to the genome of a cell or substitution of one ormore nucleotides by a different nucleotide in the genome of a cell.

In some embodiments, a cell is genetically modified using a nucleasethat is targeted to one or more selected DNA sequences. Such methods maybe used to induce precise cleavage at selected sites in endogenousgenomic loci. Genetic engineering in which DNA is inserted, replaced, orremoved from a genome, e.g., at a defined location of interest, usingtargetable nucleases, may be referred to as “genome editing”. Examplesof such nucleases include zinc-finger nucleases (ZFNs), Transcriptionactivator-like effector nuclease (TALENs), engineered meganucleasehoming endonucleases, and RNA directed nucleases such as CRISPR(clustered regularly interspaced short palindromic repeats)-associated(Cas) nucleases, e.g., derived from type II bacterial CRISPR/Cas systems(e.g., Cas9).

In some embodiments the nuclease comprises a DNA cleavage domain and aDNA binding domain (DBD) that targets the nuclease to a particular DNAsequence, thereby allowing the nuclease to be used to engineer genomicalterations in a sequence-specific manner. The DNA cleavage domain maycreate a double-stranded break (DSB) or nick at or near the sequence towhich it is targeted. ZFNs comprise DBDs selected or designed based onDBDs of zinc finger (ZF) proteins. DBDs of ZF proteins bind DNA in asequence-specific manner through one or more zinc fingers, which areregions of amino acid sequence whose structure is stabilized throughcoordination of a zinc ion. TALENs comprise DBDs selected or designedbased on DBDs of transcription activator-like (TAL) effectors (TALEs) ofXanthomonas spp. ZFN or TALEN dimers induce targeted DNA DSBs thatstimulate DNA damage response pathways. The binding specificity of thedesigned zinc-finger domain directs the ZFN to a specific genomic site.TALEs contain multiple 33-35-amino-acid repeat domains, each of whichrecognizes a single base pair. Like ZFNs, TALENs induce targeted DSBsthat activate DNA damage response pathways and enable customalterations. The DNA cleavage domain of an engineered site-specificnuclease may comprise a catalytic domain from a naturally occurringendonuclease such as the Fok1 endonuclease or a variant thereof. In someembodiments Fok1 cleavage domain variants with mutations designed toimprove cleavage specificity and/or cleavage activity may be used (see,e.g., Guo, J., et al. (2010) Journal of Molecular Biology 400 (1):96-107; Doyon, Y., et al., (2011) Nature Methods 8: 74-79. Meganucleasesare sequence-specific endonucleases characterized by a large recognitionsite (double-stranded DNA sequences of 12 to about 40 base pairs). Thesite generally occurs no more than once in a given genome. Thespecificity of a meganuclease can be changed by introducing changessequence of the nuclease (e.g., in the DNA binding domain) and thenselecting functional enzymes capable of cleaving variants of the naturalrecognition site or by associating or fusing protein domains fromdifferent nucleases.

In some embodiments, an RNA directed nuclease may be used to performgenome editing. For example, the use of CRISPR/Cas-based systems iscontemplated. In some embodiments a Cas nuclease, such as Cas9 (e.g.,Cas9 of Streptococcus pyogenes, Streptococcus thermophiles, or Neisseriameningiditis, or a variant thereof), is introduced into cells along witha guide RNA comprising a sequence complementary to a sequence ofinterest (the RNA is sometimes termed a single guide RNA). The region ofcomplementarity may be, e.g., about 20 nucleotides long. The Casnuclease, e.g., Cas9, is guided to a particular DNA sequence of interestby the guide RNA. The guide RNA may be engineered to havecomplementarity to a target sequence of interest in the genome, e.g., asequence in any gene or intergenic region of interest. The nucleaseactivity of the Cas protein, e.g., Cas9, cleaves the DNA, which candisable the gene, or cut it apart, allowing a different DNA sequence tobe inserted. In some embodiments multiple sgRNAs comprising sequencescomplementary to different genes, e.g., 2, 3, 4, 5, or more genes, areintroduced into the same cell sequentially or together. In someembodiments alterations in multiple genes may thereby be generated inthe same step.

In general, use of nuclease-based systems for genetic engineering, e.g.,genome editing, entails introducing a nuclease into cells andmaintaining the cells under conditions and for a time appropriate forthe nuclease to cleave the cell's DNA. In the case of CRISP/Cas systems,a guide RNA is also introduced. The nuclease is typically introducedinto the cell by introducing a nucleic acid encoding the nuclease. Thenucleic acid may be operably linked to a promoter capable of directingexpression in the cell and may be introduced into the cell in a plasmidor other vector. In some embodiments mRNA encoding the nuclease may beintroduced. In some embodiments the nuclease itself may be introduced.sgRNA may be introduced directly (by methods such as transfection) or byexpressing it from a nucleic acid construct such as an expressionvector. In some embodiments a sgRNA and Cas protein are expressed from asingle expression vector that has been introduced into the cell or, insome embodiments, from different expression vectors. In some embodimentsmultiple sgRNAs comprising sequences complementary to different genes,e.g., 2, 3, 4, 5, or more genes, are introduced into the same cellindividually or together as RNA or by introducing one or more nucleicacid constructs encoding the sgRNAs into the cell for intracellulartranscription.

Upon cleavage by a nuclease, a target locus (e.g., in the genome of acell) may undergo one of two major pathways for DNA damage repair,namely non-homologous end joining (NHEJ) or homology-directed repair(HDR). In the absence of a suitable repair template comprisingsufficient homology to the sequences flanking the cleavage site tostimulate HDR (see discussion below), DSBs are re-ligated through NHEJ,which can result in an insertion or deletion. NHEJ can be used, forexample, to engineer gene knockouts or generate proteins with alteredactivity. For example, an insertion or deletion in an exon can lead to aframeshift mutation or premature stop codon. Two or more DSBs can begenerated in order to produce larger deletions in the genome.

In some embodiments a nucleic acid (e.g., a plasmid or linear DNA)comprising a sequence of interest to be inserted into the genome at thelocation of cleavage is introduced into a cell in addition to anuclease. In some embodiments a sequence of interest is inserted into agene. The sequence of interest may at least in part replace the gene. Insome embodiments the nucleic acid comprises sequences that arehomologous to the sequences flanking the cleavage site, so thathomology-directed repair is stimulated. In some embodiments the nucleicacid contains a desired alteration as compared to a sequence present inthe cell's genome at or near the site of cleavage. A nucleic acidcomprising a sequence to be at least in part introduced into the genome,e.g., a nucleic acid sequence comprising homologous sequence(s) and adesired alteration may be referred to as a “donor sequence”. The donorsequence may become at least in part physically into integrated thegenome at the site of a break or may be used as a template for repair ofthe break, resulting in the introduction of all or part of thenucleotide sequence present in the donor into the genome of the cell.Thus, a sequence in a cell's genome can be altered and, in certainembodiments, can be converted into a sequence present in a donor nucleicacid. In some embodiments the donor sequence may be contained in acircular DNA (e.g. a plasmid), a linear double-stranded DNA (e.g., alinearized plasmid or a PCR product), or single-stranded DNA, e.g., asingle-stranded oligonucleotide. In some embodiments the donor sequencehas between about 10-25 bp and about 50-100 bp of homology to eitherside or each side of the target site in the genome. In some embodimentsa longer homologous sequence may be used, e.g., between about 100-500 bpup to about 1-2 kB, or more. In some embodiments an alteration isintroduced into one allele of a gene. In some embodiments a firstalteration is introduced into one allele of a gene, and a differentalteration is introduced into the other allele. In some embodiments thesame alteration is introduced into both alleles. In some embodiments twoalleles or target sites (or more) may be genetically modified in asingle step. In some embodiments two alleles or target sites (or more)may be genetically modified in separate steps.

Methods of designing, generating and using ZFNs and/or TALENs aredescribed in, e.g., WO2011097036; Urnov, F D, et al., Nature ReviewsGenetics (2010), 11: 636-646; Miller J C, et al., Nat Biotechnol. (2011)29(2):143-8; Cermak, T., et al. Nucleic Acids Research (2011) 39 (12):e82, Sanjana, N. E. et al. A transcription activator-like effectortoolbox for genome engineering. Nat Protoc 7, 171-192 (2012) andreferences in any of the foregoing. ZFN, TALEN, and CRISPR/Cas-basedmethods for genome engineering are reviewed in Gaj, T., et al., TrendsBiotechnol. 2013 July; 31(7):397-405. Epub 2013 May 9. Use of CRISPR/Cassystems in genome engineering is described in, e.g., Cong L, et al.Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-23; Mali P, et al., RNA-guided human genome engineeringvia Cas9. Science. 2013; 339(6121):823-6; Wang, H. et al. One-stepgeneration of mice carrying mutations in multiple genes byCRISPR/Cas-mediated genome engineering. Cell 153, 910-918 (2013); Ran,F. A. et al. Double Nicking by RNA-Guided CRISPR Cas9 for EnhancedGenome Editing Specificity. Cell 154, 1380-1389 (2013); Mali, P., etal., Nat Methods. 2013; 10(10):957-63; Ran, F A, Nat Protoc. 2013;8(11):2281-308). In some embodiments a nuclease that cleaves only onestrand of dsDNA (a nickase) may be used to stimulate HDR withoutactivating the NHEJ repair pathway. Nickases may be created byinactivating the catalytic activity of one nuclease monomer in the ZFNor TALEN dimer required for double stranded cleavage or inactivating acatalytic domain of a Cas protein. For example, mutations of one of thecatalytic residues (D10 in the RuvC nuclease domain and H840 in the HNHnuclease domain), e.g., to alanines (D10A, H840A) convert Cas9 into DNAnickases.

In some embodiments, a CRISP/Cas based system may be used to modulategene expression. For example, coexpression of a guide RNA with acatalytically inactive Cas9 lacking endonuclease activity generates aDNA recognition complex that can specifically interfere withtranscriptional elongation, RNA polymerase binding, or transcriptionfactor binding. This system, sometimes referred to CRISPR interference(CRISPRi), can efficiently repress expression of targeted genes inmammalian cells (Qi, S., et al., Cell. 2013; 152(5):1173-83; Larson, MH, et al., Nat Protoc. 2013; 8(10:2180-96). By attaching any of avariety of effector domains to a catalytically inactive Cas9 one cancreate a chimeric Cas9 protein that can be used to achievesequence-specific control over gene expression and/or DNA modification.Suitable effector domains include, e.g., a transcriptional activationdomain (such as those comprising the VP16 transactivation domain, e.g.,VP64), a transcriptional coactivation domain, a transcriptionalinhibitory or co-inhibitory domain, a protein-protein interactiondomain, an enzymatic domain, etc. A guide RNA guides the chimeric Cas9protein to a site of interest in the genome (e.g., in or near anexpression control element such as a promoter), whereby the effectordomain exerts an effect such as activating or inhibiting transcriptionalactivity (see, e.g., Gilbert L A, et al., Cell. 2013; 154(2):442-51;Maeder M L, et al., Nat Methods. 2013; 10(10):977-9). Appropriateeffector domains may be any of those present in naturally occurringproteins that are capable of performing the function of interest (e.g.,inhibiting or activating transcription).

Cells that have been subjected to a genetic engineering process may beselected or analyzed to identify or isolate those that express a desiredrecombinant gene product or lack expression of an endogenous gene thathas been disabled via genetic engineering or have any desired geneticalteration. For example, in some embodiments the donor sequence orvector used to deliver the donor sequence may comprise a selectablemarker, which may be used to select cells that have incorporated atleast a portion of the donor sequence comprising the selectable markerinto their genome. In some embodiments selection is not used. In someembodiments cells may be screened, e.g., by Southern blot to identifythose cells or clones that have a desired genetic alteration. Ifdesired, cells may be tested for expression level or activity of arecombinant gene product or endogenous gene product or for one or morefunctional properties associated with or conferred by a recombinant orendogenous gene product, or any other criteria of interest. Suitablemethods of analysis are known to those of ordinary skill in the art andinclude, e.g., Western blot, flow cytometry, FACS, immunofluorescencemicroscopy, ELISA assays, affinity-based methods in which cells arecontacted with an agent capable of binding to a protein of interest thatlabels or retains cells that express the protein, etc. Functional assaysmay be selected based on the identity of the recombinant gene product,endogenous gene product, and/or function or property of interest. Forexample, a functional property may be ability to bind to an antigen ofinterest or ability to exert cytotoxicity towards target cells thatexpress an antigen of interest. Cells may be analyzed, e.g., by PCR,Southern blotting, or sequencing, to determine the number of insertedDNA sequences, their location, and/or to determine whether desiredgenomic alterations have occurred. One or more cells that have desiredalteration(s), expression level, and/or functional properties may beidentified, propagated, expanded. The cells or their descendants may beused to generate a cell line, subjected to sortagging, and/or stored forfuture use.

In some embodiments, a genetically engineered cell comprises anexogenous DNA inserted into its genome. In some embodiments theexogenous DNA comprises a sequence that encodes a polypeptide ornoncoding RNA. In some embodiments the DNA comprises a cDNA. An insertedexogenous DNA may encode one or more RNAs or polypeptides. In someembodiments, the genome comprises a single copy of an exogenous DNA. Insome embodiments the genome comprises no more than 2, 3, or 4 copies. Acell may have 1, 2, 3, 4 or more distinct DNA sequences inserted intoits genome. A sequence that encodes an RNA or polypeptide may beoperably linked to appropriate expression control elements, e.g., atleast a promoter, capable of directing transcription of the sequence.The expression control elements are typically part of the exogenous DNAintegrated into the genome. Expression of a recombinant gene product maybe constitutive or conditional (e.g., inducible, repressible, cell-typespecific). A recombinant gene product may be any protein or RNAdescribed herein. In some embodiments the protein is a secreted protein,a transmembrane protein, or an intracellular protein. In someembodiments a cell a recombinant gene product comprises a selectablemarker e.g., an optically detectable protein such as a fluorescent orluminescent protein or a protein that confers resistance to a drug), atherapeutic protein, a cytokine, a chemokine, a costimulator, acoinhibitor, a growth factor, a receptor, or an adhesion molecule. Incertain embodiments the receptor is a chimeric antigen receptor, a Tcell receptor, a B cell receptor, a cytokine receptor, a chemokinereceptor, a costimulator receptor, an adhesion molecule, or a fusionprotein comprising two or more of the foregoing. In some embodiments acell is genetically engineered to produce one, more than one, or allsubunits of a multisubunit protein, e.g., a multisubunit cytokine orreceptor. In some embodiments a cell is genetically engineered toexpress a noncoding RNA. A noncoding RNA may be, e.g., a short hairpinRNA (which may be processed intracellularly to produce siRNA), a siRNA,a microRNA precursor (which may be processed intracellularly to producemiRNA), a miRNA, or a long noncoding RNA. In some embodiments thenoncoding RNA regulates expression of one or more genes, e.g., byaffecting transcription, processing, stability, and/or translation ofone or more pre-mRNAs or mRNAs. In some embodiments the noncoding RNAinhibits gene expression by the RNA interference (RNAi) pathway. Byexpressing an RNAi agent targeted towards a particular gene in a cell,expression of the gene can be stably inhibited. One of ordinary skill inthe art will be able to select appropriate sequences for an RNAi agentin order to inhibit expression of a gene of interest.

The sequence of a recombinant gene product may be a naturally occurringsequence or may be at least in part created by man (non-naturallyoccurring) in various embodiments. In some embodiments, the sequencecomprises or consists of a full length naturally occurring sequence(e.g., a polypeptide or RNA encoded in a eukaryotic genome) or afunctional variant or fragment thereof. In some embodiments, thesequence comprises or consists of one or more functional domains of anaturally occurring eukaryotic polypeptide. In some embodiments theexogenous DNA comprises a chimeric protein. For example, in someembodiments, the sequence comprises or consists of multiple functionaldomains of different naturally occurring eukaryotic polypeptides. Insome embodiments, the recombinant gene product comprises a sortaserecognition sequence or a sequence capable of serving as a nucleophilicacceptor sequence in a sortase-catalyzed reaction. In some embodimentsthe recombinant gene product does not comprise a sortase recognitionsequence or a sequence capable of serving as a nucleophilic acceptorsequence in a sortase-catalyzed reaction. In some embodiments, if asortase recognition sequence or a sequence capable of serving as anucleophilic acceptor sequence in a sortase-catalyzed reaction ispresent in a recombinant gene product, it is located in an intracellularor transmembrane domain or is otherwise not accessible to a sortaselocated outside the cell.

In some embodiments exogenous DNA is integrated into the genome of amammalian cell, e.g., a human cell, at a “safe harbor” locus. A “safeharbor” locus is an intragenic or extragenic region of the genome (e.g.,the human genome) that is generally able to accommodate the insertion ofDNA without causing a significant detectable effect on the phenotype ofhost cell (other than the effect, if any, caused by expression of arecombinant gene product encoded by the inserted DNA) and that permitsthe transcription of inserted DNA comprising suitable expression controlelements (e.g., a promoter). In some embodiments, a significant effectis a statistically significant change in the viability or proliferativecapacity of the cell or the ability of the cell to perform a normalbiological function. In some embodiments a safe harbor locus is theAAVSV1 (the natural integration site for the wild-type AAV on chromosome19), ROSA26, or CCR5 locus. The locations of these loci are well knownin the art. The AAVS1 site is in chromosome 19 (position 19q13.42) andintegration in the AAVS1 locus may disrupt the gene phosphatase 1regulatory subunit 12C (PPP1R12C). The human ROSA26 locus is inchromosome 3 (position 3p25.3). The human CCR5 gene is located onchromosome 3 (position 3p21.31). See US Pat. Pub. 20110239319 for adescription of additional sites that may be used as safe harbor loci,methods of identifying safe harbor loci, and methods of inserting DNAinto safe harbor loci. In some embodiments exogenous DNA is integratedinto a site located at least 100 kB away from any known proto-oncogeneor tumor suppressor gene.

In some embodiments a cell is genetically engineered to alter theexpression or sequence of an endogenous gene. For example, in someembodiments a gene whose expression is not desired is disabled in thecell. As used herein, a gene is “disabled” in a cell if its expressionis reduced to a level below that which is necessary for the cell toexhibit normal levels of one or more biological activities of the geneproduct or if the sequence of the encoded gene product is altered in away that reduces the activity of the gene product to a level below thatwhich is necessary for the cell to exhibit normal levels of one or morebiological activities of the gene product. A gene may be disabled by atleast partly deleting the gene, by introducing an insertion into thegene in an appropriate location, or by altering the gene so that theencoded gene product has reduced activity, e.g., by deleting or changingan active site residue or other functionally important residue. In someembodiments, precise alterations that introduce one or moresubstitutions, insertions, or deletions at a desired location are made.Such alterations may, for example change the sequence of an allele thatcauses or contributes to a disease to one that is not associated withthe disease or may change the specificity of a receptor. Expressioncontrol elements may be inserted or modified to increase or decreaseexpression of a selected endogenous gene. In some embodiments theendogenous gene encodes a receptor, a transmembrane protein, a secretedprotein, a costimulator, a coinhibitor, a cytokine, a chemokine, agrowth factor, or an adhesion molecule. In some embodiments anendogenous gene that is disabled may encode a gene product that mediatesimmunosuppressive extracellular signals (e.g., receptors for cytokinesthat may exert immunosuppressive effects, such as IL-10 or TGF-beta) orcontributes to T cell exhaustion. T cell exhaustion is a state of T celldysfunction that arises during many chronic infections and cancer. It isdefined by poor effector function, sustained expression of inhibitoryreceptors and a transcriptional state distinct from that of functionaleffector or memory T cells (reviewed in Wherry, E J, Nature Immunology,2011; 12(6):492-9). T cell exhaustion can be promoted by a variety ofcell surface inhibitory receptors such as PD-1, LNG-3, CD244 (2B4),CD160, TIM-3, and/or CTLA-4. In some embodiments inhibiting one or moresuch receptors, e.g., by disabling one or more genes that encode suchreceptors, may reduce the likelihood that a T cell will becomeexhausted. In some embodiments an endogenous gene that is disabled mayencode a gene product that mediates effects of a toxic substance such asa pro-apoptotic agent, a cytolytic agent, a cytotoxic drug, or a toxin.For example, the endogenous gene may encode a receptor for the toxicsubstance. Disabling the endogenous gene may reduce the susceptibilityof the cell to the substance relative to a cell in which the endogenousgene is expressed or functional.

As discussed herein, certain embodiments relate to sortagged mammalianimmune system cells that are capable of mounting an immune responsetowards target cells that are recognized by the sortagged immune systemcells. For example, certain embodiments relate to sortagged mammalianimmune system cells that comprise a binding moiety that binds to anantigen expressed by target cells. In some embodiments, such sortaggedimmune system cells have cytotoxic activity towards target cells. Thecells may, for example, be CD8+ T cells or NK cells. In some embodimentssuch cells are not genetically engineered.

In some embodiments immune system cells are genetically modified toexpress a recombinant gene product that binds to an antigen of interest,e.g., an antigen expressed by target cells. For example, in someembodiments a recombinant gene product comprises a chimeric antigenreceptor that binds to a particular antigen of interest. The cells may,in addition, be sortagged with any of the various agents describedherein. In some embodiments a recombinant gene product comprises a TCRchain that hinds to a particular antigen of interest. The TCR chain(s)may originate from a T cell with high affinity for a selected antigen.The T cell may originate from a human or from a non-human mammalfollowing immunization with the antigen or a portion thereof). Thenon-human mammal may be genetically engineered to express human TCRgenes (e.g., at least a TCR alpha and/or beta chain). T cells that bindto an antigen of interest may be identified, and their TCR genes may beisolated and, optionally, may be sequenced at least in part. Human TCRgenes with a desired affinity to a selected antigen may alternately oradditionally be identified using display technologies such as phagedisplay. In some embodiments, an endogenous gene that encodes a TCRchain may be modified by introducing one or more alterations that causethe binding specificity of the endogenous TCR to be directed to anantigen of interest. For example, at least a portion of the variabledomain of an endogenous TCR gene may be modified. In some embodimentsone or more CDRs may be modified. Introducing genes encoding TCRalphaand TCRbeta chains that have affinity for a selected target antigen intothe genome of a. T cell or modifying endogenous TCR chains to confer orincrease affinity for a selected target antigen creates a redirected Tcell that is capable of recognizing and responding to the targetantigen. In some embodiments one or more chains of an endogenous TCRgene may be disabled. Disabling an endogenous TCR may be useful, e.g.,when a cell is engineered to express a CAR or when a gene encoding anexogenous TCR chain is introduced into a site other than the site of thecorresponding endogenous TCR chain. Similar methods may be applied toTCR delta and gamma chains and/or BCR chains. Certain methods andcompositions useful for introducing desired TCR genes into a knownchromosomal locus a safe harbor locus), altering endogenous TCR genes,and/or inactivating TCR genes using zinc finger nucleases (ZFNs) aredescribed in U.S. Publication No. 20110158957.

A recombinant gene product may serve any one or more purposes describedherein. For example, a recombinant gene product may serve as a bindingmoiety, targeting moiety, therapeutic agent, detectable agent, or agentthat alters one or more properties of the cell. In some embodiments, anagent conjugated to the cell using sortase comprises a targeting moiety,and the recombinant gene product comprises a chimeric antigen receptor.In some embodiments, an agent conjugated to the cell using sortasecomprises a targeting moiety, and the recombinant gene product comprisesa protein or RNA that alters one or more properties of the cell.Altering one or more properties of the cell may comprise stimulating orinhibiting proliferation or activity of the cell, inhibiting orenhancing activity of a cell surface receptor, conferring responsivenessto an extracellular ligand. The recombinant gene product may increase orinhibit expression of one or more genes, bind to an intracellular domainof a receptor, increase or inhibit an intracellular protein-proteininteraction, increase or inhibit an intracellular protein translocation.In some embodiments, an agent conjugated to the cell using sortasecomprises a targeting moiety, and the recombinant gene product comprisesa different targeting moiety. The first and second targeting moietiesmay be the same or different. If different, they may bind to the sameantigen (e.g., to different epitopes thereof) or to different antigens.For example, the first and second targeting agents may bind to differenttumor antigens, which may both be present in a tumor. In someembodiments, an agent conjugated to the cell using sortase comprises atargeting moiety, and the recombinant gene product comprises atherapeutic protein. In some embodiments the targeting moiety targetsthe cell to a desired site of activity in the body.

In some embodiments, immune system cells that are to be administered toa subject or contacted with other cells ex vivo may be processed toreduce the likelihood that they will mount an immune response againstnon-target cells and/or to reduce the likelihood that a subject's immunesystem cells will mount an immune response towards administered cells.In some embodiments, T cells are rendered nonreactive to antigens thatare present on normal cells of a subject (alloantigens) by coculturingthem with allogeneic cells in the presence of one or more agents thatinhibit costimulation of the T cells. The nonreactive cells may bereferred to as “anergized cells”. The allogeneic cells may be obtainedfrom a subject to whom the anergized cells are to be administered. Insome embodiments the allogeneic cells are allogeneic PBMC. Costimulationcan be inhibited by, e.g., inhibiting the receipt of costimulatorysignals. This may be achieved by blocking the binding of B7 familymembers (e.g., B7-1 and/or B7-2) to their receptors on T cells usingagents such as anti-B7-1 and/or anti-B7-2 monoclonal antibodies. Theanergized cells preferably retain ability to recognize and kill targetcells but have reduced or absent ability to kill non-target cells. Insome embodiments.

In some embodiments, expression or activity of an endogenous TCR iseliminated or inhibited, which may be achieved, for example, byengineering a disruption or insertion in a gene encoding TCR α, TCR β,CD3zeta, and/or CD3epsilon. In some embodiments, the cell may also begenetically engineered to express a TCR or a CAR. In some embodiments,immune system cells in which endogenous TRC expression or activity iseliminated or inhibited lack ability to respond to TCR-mediatedstimulation but retain ability to be stimulated by contact with anantigen to which a TCR or CAR has specificity. (See Torikai, H., et al.,Blood (2012), 119(24): 5697-5705 for description of irreversibledisruption of endogenous TCR expression in CAR T cells using zinc fingernucleases targeting the constant regions of TCR α or TCR β genes. TheCAR T cells expressed a CAR comprising a moiety that binds to CD19. TheTCRnegCAR+ T cells did not respond to TCR-mediated stimulation bycross-linking CD3 with OKT3, but retained CD19 specificity, wereactivated and stimulated to proliferate by contact with CD19, andinduced cytotoxicity in CD19+ leukemic cells.) Such an approach may beused, e.g., to generate allogeneic antigen-specific T cells from onedonor that may be administered to multiple different recipients. In someembodiments cells are engineered to express one or more nucleic acidsencoding shRNA, siRNA, and/or miRNA molecules to down-regulateexpression of an endogenous gene encoding TCR α, TCR β, CD3zeta, and/orCD3epsilon. In some embodiments, immune system cells in which endogenousTRC expression or activity is eliminated or inhibited may beadministered to subjects who express different major and/or minorhistocompatibility antigens than do the administered cells, with reducedlikelihood of resulting in graft-versus-host-disease (GVHD) as comparedwith administration of control cells that express a functionalendogenous TCR.

In some embodiments cells, e.g., cells to be administered to a subject,are engineered to eliminate or inhibit expression of one or more HLAclass I and/or II genes, e.g., genes encoding HLA-A, HLA-B, HLA-C, orencoding an alpha or beta chain of HLA-DR, HLA-DP, or HLA-DQ. In someembodiments, this is achieved by engineering a disruption or insertionin the gene or by engineering cells to express one or more nucleic acidsencoding shRNA, siRNA, and/or miRNA molecules that inhibit expression ofthe gene. In some embodiments, eliminating or inhibiting expression oractivity of HLA class I and/or II genes of cells to be administered to asubject may reduce the likelihood that endogenous immune system cells ofa subject will mount an immune response against the administered cells.

In some embodiments, cells that are engineered to lack expression of oneor more endogenous cell-surface proteins may be contacted with one moreagents (e.g., antibodies) that binds to the extracellular portion ofsuch proteins in order to remove remaining cells (if any) that expressthe protein at their surface. The binding agent may be attached to asupport, such as magnetic beads, which retains any cells that expressthe endogenous protein at their surface For example, cells engineered tolack expression of the TCR may be contacted with anti-CD3 antibodies toremove remaining cells that express a TCR at their cell surface.

In some embodiments a cell is genetically engineered to comprise a genethat encodes an inducible suicide gene. A suicide gene is a gene thatencodes a gene product that causes a cell that contains the suicide genegene to die following induction of expression of the gene or inductionof activity of the gene product encoded by the gene. In certainembodiments at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, ormore of the cells have died within a defined time period followinginduction of expression or activity of the gene or induction of activityof the gene product, The defined time period may be, e.g., about 24-72hours. In some embodiments, the inducible suicide gene is expressed onlyin the presence of an inducer, which may be contacted with the cells exvivo or administered to a subject to whom cells comprising the suicidegene have been administered. In some embodiments, the inducible suicidegene may be expressed in the absence of an inducer, but the resultinggene product is inactive in the absence of the inducer. Administrationof the inducer to a subject comprising cells that harbor an induciblesuicide gene causes induction of the suicide gene, resulting in death orat least reduced proliferation of cells that harbor the suicide gene. Insome embodiments the protein encoded by the suicide gene has one, morethan one, or all of the following properties: non-immunogenic (at leastin humans or other mammalian subjects to whom a cell expressing thesuicide gene may be administered), non-cell-cycle dependent (thetranscription of the gene and activity of the gene product are notrestricted to particular phases of the cell cycle or limited to dividingcells or inactive in quiescent cells), clinically compatible (neitherthe gene product nor the inducing agent produce unacceptable toxicity invivo), inducible by an inducing agent that has wide biodistribution sothat the inducing agent, when administered to a subject, will reach ahigh percentage of cells harboring the gene. In some embodiments, a genethat encodes at least a portion of a human protein may be used. In someembodiments an inducible suicide gene exploits chemical inducers ofdimerization (CID) (see, e.g., Amara J F, Proc Natl Acad Sci USA. 1997;94(20):10618-23; Clackson T, et al., Proc Natl Acad Sci USA. 1998,95:10437-10442; Rollins C T, et al. Proc Natl Acad Sci USA. 2000;97(13):7096-7101). According to this approach, a proapototic molecule orother inducer of cell death is modified to comprise one or more bindingsites for a CID. Binding of the CID to its target(s) causes theiroligomerization, resulting in activation of the apoptotic pathway orother cell death-inducing pathway. In some embodiments the binding sitefor a CID comprises a mutated FK506-binding protein (FKBP12) thatmediates dimerization upon binding of a small molecule ligand (e.g., aCID such as AP1510 or AP1903). In some embodiments the CID is a dimericanalog of the immunosuppressive agent FK506. In some embodiments theanalog of FK506 lacks the immunosuppressive activity of FK506. In someembodiments the CID is biologically inert at concentrations at which itis used. Fusing one or more FKBPs, e.g., FKBP12 or a mutant thereof to apro-apoptotic domain or other cell death inducing domain, results in aprotein whose activity (pro-apoptotic activity or other cell deathinducing activity) can be stimulated by a CID. If the protein isexpressed by a cell, activity of the protein can be stimulated bycontacting the cell with a CID, e.g., by culturing the cell in thepresence of a CID or administering the CID to a subject comprising thecell. Examples of useful suicide genes include genes that encodepro-apoptotic proteins, which can be modified to render their activityinducible. For example, inducible suicide genes based on caspases 1, 3,8, or 9 (Straathof K C, et al. Blood. 2005; 105(10:4247-4254), the deathreceptor Fas (Thomis D C, et al., Blood. 2001; 97(5):1249-1257) or FADDmay be used. One such gene, designated iC9, encodes a fusion proteinthat links a truncated human caspase 9 lacking the endogenous caspaserecruitment domain (CARD) with a mutated FK506-binding protein (FKBP12).In the presence of an appropriate CID, e.g., AP1903, functional activecaspase 9 is generated, leading to apoptosis (see, e.g., Fan L, et al.,(1999) Hum Gene Ther 10: 2273-2285; see also 20110286980). In general,an inducible suicide gene may be introduced into cells using standardmethods of genetic engineering. For example, a nucleic acid encoding thegene product operably linked to a promoter may be introduced in avector, e.g., a viral vector or plasmid, which is then introduced into acell. In the case of a cell that is genetically engineered to express aCAR, the nucleic acid encoding the inducible suicide gene product may insome embodiments be included in the same construct or vector, optionallyfurther including a sequence encoding a cytokine such as IL-15. Examplesof cells that genetically engineered to express a CAR, an induciblesuicide gene, and a cytokine are described in U.S. Pat. Pub. No.20130071414.

In some embodiments, two or more nucleic acid sequences that encodedifferent proteins, different noncoding RNAs, or at least one proteinand at least one noncoding RNA, are included in a single nucleic acidconstruct, which may further include one or more operably linkedexpression control elements. Without wishing to be bound by any theory,may be advantageous as it allows coexpression of multiple gene productsfrom a single exogenous DNA. In some embodiments, transcription of eachnucleic acid sequence may be directed by a promoter operably linkedthereto, resulting in two or more separate RNAs. In some embodiments, abidirectional promoter may be used to direct transcription of twoseparate RNAs. In some embodiments, a single promoter directs expressionof an RNA that encodes multiple (e.g., two, three, four, or more)polypeptides. Translation of multiple polypeptides from one RNA can beachieved by using self-cleaving peptides or internal ribosome entrysites (IRESs). The sequences that encode the polypeptides may have aninternal ribosome entry site (IRES) located between them or a sequencethat encodes a “self-cleaving peptide” such as a 2A peptide. When IRESelements are included between multiple open reading frames (ORFs), thefirst ORF is translated by the typical cap-dependent mechanism, whilethe rest are translated through a cap-independent mechanism(Martinez-Salas, E. Curr Opin Biotechnol. 1999; 10: 458-464; Hellen C U,Sarnow P, Genes & Development, 2001; 15: 1593-1612). Since the genes aretranslated independently, the relative expression of different genes canbe adjusted, if desired, by varying the strength of the IRES locatedupstream of each ORF. IRESs are found in a variety of different viral(e.g., picornavirus) and eukaryotic mRNAs. For example, IRESes are foundin entero- and rhinoviruses, cardio- and aphthoviruses, and hepatitis Avirus. The encephalomyocarditis virus (EMCV) IRES is among the mostwidely used IRES elements for multiple gene expression in mammaliancells. An exemplary IRES sequence comprises about the region from 260 to848 in the EMCV-R genome (Genbank: M81861). Other IRES sequences usefulfor expressing multiple open reading frames encoding differentpolypeptides from a single promoter are described in Sasaki Y, JBiotechnol. 2008; 136(3-4):103-12. IRES sequences may be identical tothose found in nature or may be modified to increase or decrease theirefficiency and thereby alter the absolute and/or relative amount of thelinked ORFs. Self-cleaving 2A peptides mediate ‘ribosomal skipping’between the proline and glycine residues and inhibit peptide bondformation without affecting downstream translation. These peptides allowmultiple proteins to be encoded as polyproteins, which dissociate intocomponent proteins upon translation. Sequences linked by 2A peptides areexpressed in a single open reading frame (ORF) and “self-cleavage”occurs co-translationally to produce separate polypeptides. Use of theterm “self-cleaving” in reference to 2A peptides is common in the artand is not intended to imply a proteolytic cleavage reaction.Multicistronic vectors comprising 2A peptides between sequences thatencode proteins are reviewed in Szymczak A L, et al. 2005; 5:627-638).Self-cleaving peptides are found in members of the Picornaviridae virusfamily, including aphthoviruses such as foot-and-mouth disease virus(FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) andporcine teschovirus-1 (PTV-1) and cardioviruses such as Theilovirus(e.g., Theiler's murine encephalomyelitis) and encephalomyocarditisviruses (Donnelly, M L, et al., J. Gen. Virol., 2001; 82: 1027-1041;Ryan, M D, et al., J. Gen. Virol., 2001; 72: 2727-2732; DeFelipe, P., etal., Trends Biotechnol. 2006; 24(2):68-75). The 2A peptides derived fromFMDV, ERAV, PTV-1, and TaV are sometimes referred to as “F2A”, “E2A”,“P2A”, and “T2A”, respectively. Aphthovirus 2A polypeptides contain aDx1Ex2NPG sequence (SEQ ID NO: 5), where x1 is often valine orisoleucine. An exemplary 2A sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ IDNO: 6) from FMDV, where underlined residues are conserved in many 2Apeptides. In some embodiments a spacer sequence such as GSG or SGSG maybe included ahead of a 2A sequence. In some embodiments a proteasecleavage site may additionally be included, such as a cleavage site forfurin (RAKR). A cell may be genetically engineered to express any two ormore recombinant gene products using any of the above approaches. Insome embodiments two or more subunits of a multisubunit protein areexpressed. In some embodiments, any two or more of the followingrecombinant gene products are expressed: a receptor, a cytokine, acostimulator, a coinhibitor, a suicide protein. The receptor may be anantigen receptor (e.g., a chimeric antigen receptor), a cytokinereceptor, a costimulatory or coinhibitory receptor. All differentcombinations of products to be expressed and methods of achievingexpression of multiple products are encompassed. In some embodiments,for example, a CAR and a suicide protein, a CAR and a cytokine, or aCAR, a cytokine, and a suicide protein may be translated from a singleRNA encoded by an exogenous DNA inserted into the genome of a cell.

V. Click Chemistry

In some embodiments an agent conjugated or to be conjugated to aeukaryotic cell, e.g., a mammalian cell, may comprise a click chemistryhandle. For example, A¹ in a sortase substrate described above maycomprise a click chemistry handle. Click chemistry is a chemicalphilosophy introduced by Sharpless in 2001 and describes chemistrytailored to generate substances quickly and reliably by joining smallunits together (see, e.g., Kolb, Finn and Sharpless Angewandte ChemieInternational Edition (2001) 40: 2004-2021; Evans, Australian Journal ofChemistry (2007) 60: 384-395). Additional exemplary click chemistryhandles, reaction conditions, and associated methods useful according toaspects of this invention are described in Joerg Lahann, Click Chemistryfor Biotechnology and Materials Science, 2009, John Wiley & Sons Ltd,ISBN 978-0-470-69970-6. In some embodiments, a click chemistry handle isdescribed in any of the references herein and/or in Table 1 or Table 2.For example, a click chemistry handle may comprise or consist of aterminal alkyne, azide, strained alkyne, diene, dieneophile,alkoxyamine, carbonyl, phosphine, hydrazide, thiol, or alkene moiety.

Two entities, e.g., two proteins, each comprising a click chemistryhandle (e.g., a first protein comprising a click chemistry handleproviding a nucleophilic (Nu) group and a second protein comprising anelectrophilic (E) group that can react with the Nu group of the firstclick chemistry handle) can be covalently conjugated under clickchemistry reaction conditions. The installation of a click chemistryhandle on a protein confers click chemistry reactivity to the protein.In some embodiments a sortase-mediated conjugation is used to install afirst click chemistry handle on a polypeptide expressed by a mammaliancell, and a click chemistry reaction is then used to conjugate an entitycomprising a second click chemistry handle to the first click chemistryhandle, thereby conjugating the entity to the polypeptide and thusattaching it to the cell. In general, the second click chemistry handlemay be located at any position of the entity. In some embodiments theentity comprising the second click chemistry handle is a polypeptide. Insome embodiments the second click chemistry handle may be at theC-terminus or the N-terminus of the polypeptide or may be attached to aside chain at or near the C-terminus or N-terminus. The second clickchemistry handle may be incorporated into the entity, e.g., polypeptideusing sortase or other methods. Methods of installing click chemistryhandles on polypeptides are described in PCT/US2012/044584. In someembodiments the use of click chemistry allows two proteins to beconjugated at their respective N-termini, generating an N—N conjugatedchimeric protein. For example, the N-terminus of a non-geneticallyengineered polypeptide expressed by a mammalian cell may be modifiedusing sortase to install a first click chemistry handle, as describedherein. A polypeptide having a compatible click chemistry handleinstalled at or near its N-terminus may then be conjugated via clickchemistry to the first click chemistry handle.

Click chemistry should be modular, wide in scope, give high chemicalyields, generate inoffensive byproducts, be stereospecific, bephysiologically stable, exhibit a large thermodynamic driving force(e.g., >84 kJ/mol to favor a reaction with a single reaction product),and/or have high atom economy. Several reactions have been identifiedwhich fit this concept:

(1) The Huisgen 1,3-dipolar cycloaddition (e.g., the Cu(I)-catalyzedstepwise variant, often referred to simply as the “click reaction”; see,e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064).Copper and ruthenium are the commonly used catalysts in the reaction.The use of copper as a catalyst results in the formation of1,4-regioisomer whereas ruthenium results in formation of the1,5-regioisomer;

(2) Other cycloaddition reactions, such as the Diels-Alder reaction;

(3) Nucleophilic addition to small strained rings like epoxides andaziridines;

(4) Nucleophilic addition to activated carbonyl groups; and

(5) Addition reactions to carbon-carbon double or triple bonds.

For two proteins to be conjugated via click chemistry, the clickchemistry handles of the proteins have to be reactive with each other,for example, in that the reactive moiety of one of the click chemistryhandles can react with the reactive moiety of the second click chemistryhandle to form a covalent bond. Such reactive pairs of click chemistryhandles are well known to those of skill in the art and include, but arenot limited to those described in Table I:

TABLE 1 Exemplary click chemistry handles and reactions.

In some embodiments R, R₁ or R₂ in click chemistry handles and reactionsabove is a non-genetically engineered polypeptide -[Xaa]_(y)-TRS-PRTaccording to Formula I above expressed by a living mammalian cell,wherein the polypeptide has been modified by sortase-catalyzedconjugation of a click chemistry handle thereto, and the other of R, R₁or R₂ is a moiety to be conjugated to the click chemistry handle of themodified polypeptide.

In some embodiments, click chemistry handles are used that can react toform covalent bonds in the absence of a metal catalyst. Such clickchemistry handles are well known to those of skill in the art andinclude the click chemistry handles described in Becer, Hoogenboom, andSchubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition,Angewandte Chemie International Edition (2009) 48: 4900-4908.

TABLE 2 exemplary click chemistry handles and reactions. Reagent AReagent B Mechanism Notes on reaction^([a]) Reference 0 azide alkyneCu-catalyzed [3 + 2] 2 h at 60° C. in H₂O  [9] azide-alkynecycloaddition (CuAAC) 1 azide cyclooctyne strain-promoted [3 + 2] 1 h atRT [6-8, azide-alkyne cycloaddition 10, 11] (SPAAC) 2 azide activated[3 + 2] Huisgen 4 h at 50° C. [12] alkyne cycloaddition 3 azideelectron- [3 + 2] cycloaddition 12 h at RT in H₂O [13] deficient alkyne4 azide aryne [3 + 2] cycloaddition 4 h at RT in THF [14, 15] with crownether or 24 h at RT in CH₃CN 5 tetrazine alkene Diels-Alder retro- 40min at 25° C. [36-38] [4 + 2] cycloaddition (100% yield) N₂ is the onlyby-product 6 tetrazole alkene 1,3-dipolar cycloaddition few min UVradiation and [39, 40] (photoclick) then overnight at 4° C. 7dithioester diene hetero-Diels-Alder 10 min at RT [43] cycloaddition 8anthracene maleimide [4 + 2] Diels-Alder 2 days at reflux in toluene[41] reaction 9 thiol alkene radical addition 30 min UV (quantitativeconv.) [19-23] (thio click) or 24 h UV irradiation (>96%) 10 thiol enoneMichael addition 24 h at RT in CH₃CN [27] 11 thiol maleimide Michaeladdition 1 h at 40° C. in THF or [24-26] 16 h at RT in dioxane 12 thiolpara-fluoro nucleophilic substitution overnight at RT in DMF or [32] 60min at 40° C. in DMF 13 amine para-fluoro nucleophilic substitution 20min MW at 95° C. in [30] NMP as solvent ^([a])RT = room temperature, DMF= N,N-dimethylformamide, NMP = N-methylpyrolidone, THF =tetrahydrofuran, CH₃CN = acetonitrile.From Becer, Hoogenboom, and Schubert, click Chemistry beyondMetal-Catalyzed Cycloaddition, Angewandte Chemie International Edition(2009) 48: 4900-4908.

Additional click chemistry handles suitable for use in methods ofconjugation described herein are well known to those of skill in theart, and such click chemistry handles include, but are not limited to,the click chemistry reaction partners, groups, and handles described inPCT/US2012/044584 and references therein, which references areincorporated herein by reference for click chemistry handles andmethodology.

In some embodiments eukaryotic cells, e.g., mammalian cells are modifiedby using sortase to conjugate a moiety comprising a first clickchemistry handle to a polypeptide expressed by the cells, wherein thepolypeptide is not genetically engineered to comprise an extracellularsortase recognition motif or extracellular glycine, e.g.,non-genetically engineered eukaryotic, e.g., mammalian, cells. Once aclick chemistry handle has been installed using sortase, the cells maybe further modified by click chemistry mediated attachment of anyavailable entity comprising a compatible click chemistry handle, withoutneed to modify the entity to incorporate a sortase recognition motif.This approach may facilitate rapid conjugation of diverse entities ontocells. In some embodiments a population of cells that have been modifiedby using sortase to conjugate a moiety comprising a first clickchemistry handle to a polypeptide expressed by the cells may be dividedinto two or more aliquots. The number of aliquots and number of cellsper aliquot may be selected in any convenient manner. In someembodiments aliquots comprise at least 10³, 10⁴, 10⁵, 10⁶, or 10⁷ cells.In some embodiments the number of aliquots is between 2 and 1,000. Oneor more aliquots may be stored for future use. In some embodiments twoor more different moieties, each comprising a second click chemistryhandle that is compatible with the first click chemistry handle areconjugated to cells of two or more different aliquots, to produce two ormore populations of modified mammalian cells having the differentmoieties conjugated thereto. Cells of different aliquots, or portionsthereof, having different entities conjugated thereto, may besubsequently combined.

VI. Sortagged Mammalian Cells and Uses Thereof

Without limiting the invention, this section discusses certain proteins,nucleic acids, lipids, small molecules, and other entities of interestin the context of the present disclosure, certain methods of preparingor using sortagged mammalian cells, and related compositions. Entititesdescribed herein may be used for various purposes. For example, invarious embodiments proteins, nucleic acids, lipids, small molecules,and sugars may be conjugated to mammalian cells using sortase,conjugated to each other using sortase, used in cell culture (e.g., toexpand, stimulate, or differentiate cells), and/or administered to asubject.

In some embodiments a first protein is conjugated to a second proteinusing sortase. In some embodiments the second protein is expressed by aliving mammalian cell. For example, A¹ in sortase substrate A describedabove may comprise any protein. In general, any protein or polypeptidethat comprises or is modified to comprise an appropriately positionedsortase recognition motif can be conjugated to a living mammalian cell.In some embodiments a protein is modified by conjugating an agent to itusing sortase, and the resulting protein is conjugated to a mammaliancell using sortase. In some embodiments two or more polypeptides areconjugated using sortase, and the resulting protein is conjugated to amammalian cell using sortase. In some embodiments a polypeptide may beextended to include a sortase recognition motif at or near itsC-terminus and/or to include one or more N-terminal glycines or otherappropriate nucleophilic acceptor sequence to allow it to participate ina sortase-catalyzed reaction.

In some embodiments a protein is an enzyme, e.g., an enzyme that plays arole in metabolism or other physiological processes in a mammal. In someembodiments a protein, is characterized in that deficiency of theprotein underlies a disease that affects a mammal. In some embodiments aprotein is an enzyme that plays a role in carbohydrate metabolism, aminoacid metabolism, organic acid metabolism, porphyrin metabolism, purineor pyrimidine metabolism, and/or lysosomal storage. Deficiencies ofenzymes or other proteins can lead to a variety of diseases, e.g.,diseases associated with defects in carbohydrate metabolism, amino acidmetabolism, organic acid metabolism, purine or pyrimidine metabolism,lysosomal storage disorders, and blood clotting, among others. Examplesinclude the following (name of deficient enzyme or other protein isindicated in parentheses if not part of name of disease):glucose-6-phosphate dehydrogenase deficiency, alpha-1 antitrypsindeficiency, phenylketonuria (deficiency in phenylalanine hydroxylase),Fabry disease (alpha galactosidase A deficiency), Gaucher disease(glucocerebrosidase deficiency), Pompe disease (acid alpha-glucosidasedeficiency), adenosine deaminase deficiency, mucopolysaccharidoses suchas MPSI (alpha-L-iduronidase deficiency), MPSII (iduronate-2-sulfatasedeficiency), MPSIIIA (heparan sulfamidase deficiency), MPSVI(N-acetylgalactosamine-4-sulfatase deficiency), hemophilia (variouscoagulation factors), hereditary angioedema (C1 esterase inhibitordeficiency); hypophosphatasia (tissue-nonspecific isozyme of alkalinephosphatase (TNSALP). In some embodiments the enzyme is one whereinexogenous administration of the enzyme at least in part alleviates thedisease. In some embodiments the enzyme is one that is normally presentin the blood. In some embodiments the enzyme may normally be produced bycells in the liver or kidneys and secreted into the blood. In someembodiments the enzyme acts on a substrate whose increased presence oraccumulation in the blood may contribute to a disease or which may betransported by the blood to a site in the body where it contributes to adiseases. In some embodiments a deficiency is due to an inheritedmutation. In some embodiments a deficiency may be transient, e.g., itmay be due at least in part to excessive consumption, degradation, orloss of the enzyme (e.g., due to bleeding), or due at least in part totoxicity (e.g., exposure to a toxin that inactivates the enzyme orincreases requirement for the enzyme). In some embodiments a disease,e.g., an enzyme deficiency disease, is a rare disease or orphan diseaseas defined in the US or as defined in the relevant jurisdiction where apatient is treated.

In some embodiments an enzyme conjugated to mammalian cells iscatalytically active. In some embodiments the enzyme is in acatalytically inactive form (e.g., a zymogen) and is cleaved in vivo(after administration of the cells to a subject) to generate an activeform. Such cleavage may be catalyzed by an endogenous protease. In someembodiments the enzyme may be released from cells in vivo. Such releasemay occur via a proteolytic cleavage that, in some embodiments, alsoactivates the enzyme. It will be understood that an enzyme used to treatan enzyme deficiency need not be the same as the deficient enzyme solong as it provides the required enzymatic activity, e.g., at least theenzymatic activity necessary to treat the disease.

In some embodiments an agent comprises both (i) a therapeutically activedomain, e.g., an enzyme, small molecule, therapeutic protein,therapeutic antibody, and (ii) a targeting domain, wherein the targetingdomain targets the cells and/or agent to a site in the body where thetherapeutic activity is desired. The targeting domain binds to a targetpresent at such site. Any targeting domain may be used, e.g., anantibody. In some embodiments the agent may be released from cellsurfaces after administration and the released agent, comprising both atargeting domain and a therapeutic domain, accumulates at a site ofdisease. The site may be any organ or tissue, e.g., any organ or tissuewhere the disease causes destruction, degradation, or symptoms. Forexample, an agent may be targeted to respiratory tract (e.g., lung),bone, kidney, liver, pancreas, skin, cardiovascular system (e.g.,heart), smooth or skeletal muscle, gastrointestinal tract, eye, bloodvessel surfaces, etc.

In some embodiments an agent conjugated to mammalian cells, e.g.,hematopoietic cells, e.g., red blood cells, comprises a moiety thatmodulates blood coagulation or breakdown of blood clots (fibrinolysis).In some embodiments the moiety promotes blood coagulation, e.g., themoiety may be a protein that participates in the coagulation pathway,e.g., a coagulation factor (e.g., factor VII, VIIa, VIII or IX). In someembodiments, mammalian cells conjugated with a moiety that promotesblood coagulation, such as a coagulation factor, may be administered toa subject to speed blood clotting in order to promote cessation of bloodloss from a damaged vessel (e.g., a subject who has experienced aphysical injury). In some embodiments cells conjugated with an agentthat promotes blood coagulation may be administered prophylactically,e.g., to a subject with a defect in blood coagulation (e.g., hemophilia)with an aim of preventing excessive blood loss in case the subject isinjured, to a subject who is to undergo surgery that presents a risk ofsignificant blood loss, or to a subject who has received an excessiveamount of an anticoagulant. In some embodiments the moiety inhibitscoagulation and/or promotes fibrinolysis, e.g., the moiety may comprisea coagulation pathway regulator, heparin, plasmin, tissue plasminogenactivator, streptokinase, urokinase, or a variant of any of these. Insome embodiments cells conjugated with a moiety that inhibitscoagulation and/or promotes fibrinolysis may be administered to asubject at risk of blood clot formation. For example, the subject mayhave an arrhythmia such as atrial fibrillation and/or a history ofpathologic coagulation (e.g., embolism, thrombophlebitis, ischemicstroke), etc. The moiety that inhibits coagulation and/or promotesfibrinolysis may comprise Protein C, antithrombin, tissue factor pathwayinhibitor, or plasmin.

In some embodiments, a protein comprises a receptor or receptor fragment(e.g., at least a portion of an extracellular domain). In someembodiments the receptor is a cytokine receptor, growth factor receptor,interleukin receptor, or chemokine receptor. In certain embodiments agrowth factor receptor is a TNFα receptor (e.g., Type I TNF-α receptor),VEGF receptor, EGF receptor, PDGF receptor, IGF receptor, NGF receptor,or FGF receptor. In some embodiments the protein comprises at least asufficient portion of a receptor to bind to a natural ligand of thereceptor. In some embodiments the protein is capable of acting as adecoy receptor, i.e., a receptor that binds a ligand and therebyinhibits the ligand from binding to its normal receptor. In someembodiments cells conjugated with a decoy receptor that binds to anatural ligand are administered to a subject in order to inhibitactivity of the natural ligand. In some embodiments cells conjugatedwith a decoy receptor that binds to a natural ligand are administered toa subject suffering from a disease that can be effectively treated byadministration of a decoy receptor or other inhibitor of the ligandand/or by administration of an inhibitor of a natural receptor for theligand, e.g., a disease that is at least in part caused or exacerbatedby a natural ligand of the receptor. For example, a protein comprising asoluble TNF receptor, e.g., etanercept may be used in treatment of avariety of inflammatory and autoimmune diseases such as rheumatoidarthritis, psoriasis, ankylosing spondylitis, and Behcet's disease.

In certain embodiments, a protein comprises urate oxidase. Urate oxidasecan be formulated as a protein drug (rasburicase) for the treatment ofacute hyperuricemia, e.g., in patients receiving chemotherapy. In someembodiments cells having urate oxidase conjugated thereto may beadministered to a subject in need of treatment of acute hyperuricemia,e.g., a patient receiving chemotherapy). Acute hyperuricemia may occuras a feature of tumor lysis syndrome in patients receiving chemotherapy,e.g., for hematologic cancers such as leukemias and lymphomas. In someembodiments cells having urate oxidase conjugated thereto may beadministered to a subject in need of treatment of chronic hyperuricemia,e.g., a patient with gout, e.g., gout that is refractory to othertreatments.

In some embodiments a protein is a cytokine. In some embodiments acytokine is an interleukin (IL) e.g., any of IL-1-IL-38. In someembodiments a protein is a four-helix bundle protein, e.g., a four-helixbundle cytokine. In some embodiments a four-helix bundle cytokine is amember of the IL-2 subfamily, the interferon (IFN) subfamily, or theIL-10 subfamily. Exemplary four-helix bundle cytokines include, e.g.,certain interferons (e.g., a type I interferon, e.g., IFN-α),interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12), andcolony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF). The IFN can be,e.g., interferon alpha 2a or interferon alpha 2b. See, e.g., Mott H Rand Campbell I D., Curr Opin Struct Biol. 1995, 5(1):114-21; Chaiken IM, Williams W V, Trends Biotechnol. 1996, 14(10):369-75; Klaus W, etal., J Mol Biol., 274(4):661-75, 1997, for further discussion of certainof these cytokines. In some embodiments, the cytokine has a similarstructure to one or more of the afore-mentioned cytokines. For example,the cytokine can be an IL-6 class cytokine such as leukemia inhibitoryfactor (LIF) or oncostatin M. In some embodiments, the cytokine is onethat in nature binds to a receptor that comprises a GP130 signaltransducing subunit. Other four-helix bundle proteins include growthhormone (GH) and prolactin (PRL). In some embodiments, the protein is anerythropoiesis stimulating agent, e.g., erythropoietin (EPO), which isalso a four-helix bundle cytokine. In some embodiments, anerythropoiesis stimulating agent is an EPO variant, e.g., darbepoetinalfa, also termed novel erythropoiesis stimulating protein (NESP), whichis engineered to contain five N-linked carbohydrate chains (two morethan recombinant HuEPO). In some embodiments the cytokine is one thatstimulates differentiation, activation, survival, and/or proliferationof one or more types or subtypes of immune system cells, e.g., T cells(e.g., CD4+ helper T cells, CD8+ cytotoxic T cells, Tregs), NK cells, Bcells, DCs, monocytes, macrophages, or precursors of any of theforegoing.

In some embodiments, the protein comprises five helices. For example,the protein can be an interferon beta, e.g., interferon beta-1a orinterferon beta-1b, which (as will be appreciated) is often classifiedas a four-helix bundle cytokine.

In some embodiments a cytokine is an IL-12 family member. IL-12 familymembers are heterodimeric cytokines and include, e.g., IL-12, IL-23,IL-27 and IL-35. IL-12 and IL-23 have mainly proinflammatory properties,whereas IL-27 and IL-35 have mainly anti-inflammatory properties. IL-12and IL-23 share the β-chain p40 (IL-12β), whereas IL-27 and IL-35 sharethe β-chain Epstein-Barr virus-induced gene 3 (EBI3). IL-12 and IL-35share the α-chain p35, whereas IL-23 and IL-27 have unique α-chains.IL-12 and IL-23 are disulfide-linked heterodimers whereas IL-27 andIL-35 lack a disulfide linkage. IL-35 is produced mainly by Treg cells.IL-12, IL-23 and IL-27 are secreted by myeloid cells such as macrophagesand DCs, e.g., after stimulation by contact with specificpathogen-associated molecular patterns. IL-12 is composed of IL-12A(p35) and IL-12B (p40) subunits. IL-12 is involved in thedifferentiation of naive T cells into Th1 cells and plays an importantrole in enhancing the activities of natural killer cells and Tlymphocytes. IL-23 is a composed of p40, which is also a component ofIL-12, and p19, which is considered the IL-23 alpha subunit. IL-23 hasseveral proinflammatory effects including the capacity to stimulatenaive CD4+ T cells to differentiate into Th17 cells. IL-27 consists oftwo subunits p28 and EBI3. It acts as a differentiation factor in thegeneration of Tr1 cells and inhibits Th17 cells. IL-35 heterodimers arecomposed of EBI3 and the IL-12p35 subunit. See, e.g., Hunter, C A,Nature Reviews Immunology 5: 521-531, 2005, for discussion of certainIL-12 family members.

In some embodiments a cytokine is IL-17. In some embodiments a cytokineis IL-9, IL-10, IL-11, IL-13, IL-14, or IL-15.

In some embodiments, a protein comprises a biologically active portionof a cytokine, e.g., IL-2, IL-7, IL-12, IL-15, or IL-21. In someembodiments, a multi-subunit cytokine such as IL-12 is produced as asingle polypeptide. In some embodiments the polypeptide preserves theN-terminus of the p40 subunit of IL-12 in a sequential p40-p35 fusion.In some embodiments the polypeptide comprises a p35-p40 fusion. In someembodiments the subunits are joined by a spacer, e.g. a polypeptidelinker, in a fusion protein. Examples of bioactive fusion proteinscomprising the p35 and p40 subunits of IL-12, nucleic acids and vectorsencoding such proteins, and methods of producing the proteins aredescribed in U.S. Pat. No. 5,891,680. In some embodiments the p35 andp40 are expressed as parts of separate polypeptides that permitheterodimerization of the p35 and p40 subunits may be used.

In some embodiments, a protein comprises a biologically active variantor fragment of a co-stimulatory molecule or cell adhesion molecule,wherein the biologically variant or fragment is capable of binding to anaturally occurring receptor, ligand, or interaction partner of suchmolecule.

In some embodiments a protein is a subunit of a multisubunit protein,e.g., a multisubunit cytokine or multisubunit cytokine receptor. In someembodiments a subunit is unique to a particular cytokine or cytokinereceptor. In some embodiments a subunit is found in multiple differentcytokines or cytokine receptors. In some embodiments, two or moresubunits of a multisubunit protein may be linked to form a singlemolecule. The two or more subunits may be linked to form a singlepolypeptide, e.g., as a fusion protein, or may be linked by any suitablelinker. In some embodiments the two or more subunits may be linked usingclick chemistry. The two or more subunits may be separated from eachother by a spacer, e.g., a polypeptide spacer, which may facilitateassembly of the subunits to form the normal quaternary structure of theprotein. In some embodiments, two or more subunits of a multisubunitprotein may be individually attached to a cell or expressed by a cell.

In some embodiments a protein promotes survival, proliferation and/ordifferentiation of one or more cell types. A protein may provide anextracellular signal that is necessary or promotes survival, e.g., byinhibiting apoptosis. One of ordinary skill in the art will be aware ofcertain proteins that act as survival factors for particular cell types.Such proteins include, e.g., growth factors, cytokines, chemokines, andothers.

In some embodiments, a protein comprises a growth factor for one or morecell types. Growth factors include, e.g., members of the vascularendothelial growth factor (VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D),epidermal growth factor (EGF), insulin-like growth factor (IGF; IGF-1,IGF-2), fibroblast growth factor (FGF, e.g., FGF1-FGF22), plateletderived growth factor (PDGF), or nerve growth factor (NGF) families. Itwill be understood that the afore-mentioned protein families comprisemultiple members. Any member may be used in certain embodiments. In someembodiments a growth factor promotes survival, proliferation and/ordifferentiation of one or more hematopoietic cell types. For example, agrowth factor may be CSF1 (macrophage colony-stimulating factor), CSF2(granulocyte macrophage colony-stimulating factor, GM-CSF), or CSF3(granulocyte colony-stimulating factors, G-CSF), stem cell factor (SCF),thrombopoietin (TPO), or Flt-3 ligand. In some embodiments, mammaliancells that have a growth factor or growth factor receptor agonistconjugated thereto may be contacted with cells in vitro or administeredto a subject in order to promote proliferation of cells whoseproliferation is stimulated by such moieties.

In some embodiments a protein is a chemokine. Chemokines are a family ofsmall cytokines that have the ability to induce directed chemotaxis inresponsive cells, i.e., they are chemotactic cytokines. Proteins may beclassified as chemokines according to shared structural characteristicssuch as small size (approximately 8-10 kilodaltons in size), and thepresence of at least two cysteine residues, e.g., four cysteineresidues, in conserved locations that play an important role in their3-dimensional structure (some chemokines contain one or more additionalcysteines). The chemokine family may be divided into four subfamiliesdepending on the spacing of their first two cysteine residues: CXC,CX3C, C (or XC), and CX3C chemokines. A chemokine may be CCL1-CCL28,CXCL1-CXCL17, XCL1 or XCL2, or CXC3L1. Chemokine receptors are Gprotein-coupled receptors containing 7 transmembrane domains. Chemokinereceptors may be divided into four families depending on the type ofchemokine they bind; CXCR that bind CXC chemokines, CCR that bind CCchemokines, CX3CR1 that binds the CX3C chemokine (CX3CL1), and XCR1 thatbinds the two XC chemokines (XCL1 and XCL2). In some embodiments,mammalian cells that have a chemokine or chemokine receptor agonistconjugated thereto may be contacted with cells in vitro or administeredto a subject in order to promote migration of cells whose migration isstimulated by such moieties.

In some embodiments, a protein is a neurotrophic factor, i.e., a factorthat promotes survival, development and/or function of neural lineagecells (which term as used herein includes neural progenitor cells,neurons, and glial cells, e.g., astrocytes, oligodendrocytes,microglia). For example, in some embodiments, the protein is a factorthat promotes neurite outgrowth. In some embodiments, the protein isciliary neurotrophic factor (CNTF; a four-helix bundle protein) or ananalog thereof such as Axokine, which is a modified version of humanciliary neurotrophic factor with a 15 amino acid truncation of the Cterminus and two amino acid substitutions, which is three to five timesmore potent than CNTF in in vitro and in vivo assays and has improvedstability properties. In some embodiments, mammalian cells that have aneutrophic factor or neurotrophic factor receptor agonist conjugatedthereto may be contacted with cells in vitro or administered to asubject in order to promote survival, development, and/or function ofcells whose survival, development, and/or function is stimulated by suchmoieties.

In some embodiments a protein comprises a constant domain of an antibody(e.g., an Fc domain) that recruits Fc receptor-bearing cells, e.g.,monocytes, dendritic cells, and natural killer cells. Uponadministration of the sortagged cells to a subject, Fc receptor-bearingcells are recruited to a location to which the sortagged cell is present(e.g., a site of target cells in a tumor or site of infection). The Fcreceptor-bearing cells may further promote an immune response mounted bythe sortagged cells, may promote an immune response by endogenous immunesystem cells of the subject, or may mount their own immune responseagainst target cells. In some embodiments a protein comprises a constantdomain of an antibody (e.g., an Fc domain) that has been modified toalter (e.g., increase or decrease) one or more activities that such Fcdomain would otherwise have. The modification may, for example, alterthe ability of the Fc domain to recruit Fc receptor-bearing cells and/orfix complement. In some embodiments a protein does not comprise an Fcdomain or such domain is modified so that it does not bind to an Fcreceptor and/or fix complement.

In some embodiments a protein comprises or consists of a polypeptidethat is identical in sequence to or is a variant of a naturallyoccurring protein or polypeptide, e.g., a variant that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to anaturally occurring protein or polypeptide or fragment thereof. In someembodiments a protein has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid differences relative to a naturally occurring sequence. Insome embodiments a naturally occurring protein is a mammalian protein,e.g., of human origin. In some embodiments a protein is an antibody, anantibody fragment, or protein comprising an antigen-binding domain.

In some embodiments a protein is a chimeric protein comprising two ormore different polypeptides that are not found joined together innature. For example, a chimeric protein may comprise two or moredifferent targeting moieties or may comprise a targeting moiety and atherapeutic agent. In some embodiments the two or more polypeptides arejoined directly to each other, e.g., via peptide bond(s). In someembodiments the two or more polypeptides are joined to each other viaone or more linkers, which may comprise or consist of one or more aminoacid(s), e.g., a polypeptide. In some embodiments the two or morepolypeptides are joined in a single polypeptide chain. In someembodiments the polypeptide chain comprises one or more amino acids,e.g., a polypeptide linker, between any two portions of the polypeptide.If the polypeptide comprises multiple polypeptide linkers, they may bethe same or different in sequence. Any polypeptide can be extended,e.g., to comprise one or more additional amino acids, e.g., apolypeptide linker. In some embodiments at least two of the polypeptidesare subunits of the same protein, e.g., the same cytokine or cytokinereceptor. In some embodiments a chimeric protein may be generated byproducing a nucleic acid that encodes the chimeric protein andexpressing the nucleic acid in a suitable expression system usingstandard methods. In some embodiments sortase may be used to generate achimeric protein.

In some embodiments a protein is one that forms homodimers orheterodimers, (or homo- or heterooligomers comprising more than twosubunits, such as tetramers). In certain embodiments the homodimer,heterodimer, or oligomer structure is such that a terminus of a firstsubunit is in close proximity to a terminus of a second subunit. Forexample, an N-terminus of a first subunit is in close proximity to aC-terminus of a second subunit. In certain embodiments the homodimer,heterodimer, or oligomer structure is such that a terminus of a firstsubunit and a terminus of a second subunit are not involved ininteraction with a receptor, so that the termini can be joined via,e.g., a non-genetically encoded peptide element, without significantlyaffecting biological activity. In some embodiments, termini of twosubunits of a homodimer, heterodimer, or oligomer are conjugated viaclick chemistry using a method described herein, thereby producing adimer (or oligomer) in which at least two subunits are covalentlyjoined. For example, the neurotrophins nerve growth factor (NGF);brain-derived neurotrophic factor (BDNF); neurotrophin 3 (NT3); andneurotrophin 4 (NT4) are dimeric molecules which share approximately 50%sequence identity and exist in dimeric forms. See, e.g., Robinson R C,et al., Biochemistry. 34(13):4139-46, 1995; Robinson R C, et al.,Protein Sci. 8(12):2589-97, 1999, and references therein. In someembodiments, the dimeric protein is a cytokine, e.g., an interleukin.

In some embodiments a protein is a member of the immunoglobulinsuperfamily (IgSF). The IgSF is a group of cell surface and solubleproteins that are involved in a variety of cell processes, such asrecognition, binding, and/or adhesion. Proteins are classified asmembers of the IgSF based on characteristic shared structural featureswith immunoglobulins (antibodies), i.e., they possess a domain known asan immunoglobulin domain or fold which is found in antibodies. Membersof the IgSF include, e.g., antibodies, cell surface antigen receptors,co-receptors and costimulatory molecules of the immune system, moleculesinvolved in antigen presentation to lymphocytes, cell adhesionmolecules, certain cytokine receptors, and various intracellular muscleproteins. Ig domains contain about 70-110 amino acids and may be furthercategorized according to their size and function. Ig domains possess acharacteristic fold formed by two sheets of antiparallel beta strands.The sheets are held together by a disulfide bond. Cell surface antigenreceptors include (i) the T cell receptor (TCR), which comprises twochains, either the TCR-alpha and -beta chains or the TCR-delta and gammachains, associated with a CD3 complex; (ii) the B cell receptor (BCR),which comprises cell surface immunoglobulin associated withantigen-nonspecific signaling molecules termed Ig alpha and Ig beta.Major histocompatibility complex (MHC) proteins are ligands for TCRs.MHC class I proteins form a dimer with beta-2 microglobulin ((32M) andinteract with the TCR on cytotoxic T cells. MHC class II proteins havetwo chains (alpha and beta) that interact with the TCR on helper Tcells. MHC class I, MHC class II and β2M proteins all possess Ig domainsand therefore are members of the IgSF. MHC class I, MHC class II, andbeta-2 microglobulin function as antigen presenting molecules (APMs).Co-receptors include a variety of proteins that function together withprimary receptors to mediate a cellular event. Certain co-receptors areexpressed on the surfaces of lymphocytes and interact with MHC moleculesduring TCR or BCR engagement. The co-receptor CD4 is found on helper Tcells and the co-receptor CD8 is found on cytotoxic T cells. Aco-receptor complex including the proteins CD19, CD21, and CD89 is usedby the BCR. Certain co-receptors regulate T cell function positively(e.g., CD28) or negatively (e.g., cytotoxic T lymphocyte antigen 4(CTLA-4)) by interacting with distinct cell-surface receptors.Costimulatory or inhibitory molecules include a variety of signalingreceptors and ligands that regulate the activation, expansion andeffector functions of immune system cells. A major group of IgSFco-receptors are molecules of the CD28 family, e.g., CD28, CTLA-4,programmed cell death-1 (PD-1), the B- and T-lymphocyte attenuator(BTLA, CD272, also referred to as B and T cell associated), and theinducible T-cell costimulator (ICOS, CD278). IgSF ligands of thesemolecules include members of the B7 family, e.g., CD80 (B7-1), CD86(B7-2), ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4. Theleukocyte immunoglobulin-like receptors (LILR) are members of the IgSF.They are also known as CD85, ILTs and LIR families, and can exertimmunomodulatory effects on a wide range of immune cells.

In some embodiments, a protein is one that participates in cell-cell orcell-substrate physical interactions. For example, the protein maymediate binding of cells to each other or to the extracellular matrix(ECM). A physical interaction between cells may be between cells of thesame type or between cells of different types. In some embodiments aphysical interaction between cells or between cells and ECM may berelatively stable, such as those cell involved in establishing ormaintaining the structure or organization of tissues. Examples ofproteins that participate in cell-cell or cell-substrate physicalinteractions include, e.g., cell adhesion molecules (CAMs). Most CAMsbelong to any of four protein families: immunoglobulin (Ig) superfamily(IgSF) CAMs, integrins, the cadherin superfamily, and the selectins.Many CAMs are transmembrane proteins composed of three domains: anintracellular domain that interacts with the cytoskeleton, atransmembrane domain, and an extracellular domain that interacts withother CAMs or CAM ligands. Members of all CAM superfamilies can mediatecell-cell interactions, whereas integrins can also mediate cell-matrixinteractions. CAMs bind to either the exact same protein, known ashomophilic binding, or different proteins, called heterophilic binding.IgSF CAMs include, e.g., synaptic cell adhesion molecules, neural celladhesion molecules (NCAMs), intercellular cell adhesion molecule(ICAM-1), vascular cell adhesion molecule (VCAM-1), platelet-endothelialcell adhesion molecule (PECAM-1). The cadherin superfamily is composedof proteins that include extracellular cadherin domains (ECD). Suchproteins include cadherins, protocadherins, desmogleins, anddesmocollins, and others (see, e.g., Hulpiau P, van Roy F (2009),“Molecular evolution of the cadherin superfamily”. Int. J. Biochem. CellBiol. 41 (2): 349-69). Cadherins are Ca2+-dependent glycoproteins.Different cadherins typically exhibit different patterns of tissuedistribution, and many of these proteins are named according to tissuein which they are typically found, such as epithelial (E-cadherins),placental (P-cadherins), neural (N-cadherins), retinal (R-cadherins),brain (B-cadherins and T-cadherins) and muscle (M-cadherins). Many celltypes express multiple cadherin types.

Integrins mediate cell interactions with a variety of ligands on othercells or in the ECM such as collagen, fibrinogen, fibronectin, laminin,and vitronectin. Integrins are heterodimers, consisting of an alpha andbeta subunit. There are at least 18 alpha subunits and at least 8 betasubunits in mammals, which combine to make up at least 24 differentintegrin proteins. Many integrins bind to ligands containing an RGD orLDV tripeptide.

In some embodiments a CAM or CAM ligand is conjugated to mammalian cellsusing sortase. A CAM or CAM ligand may be used as a targeting moiety. Amammalian cell that has a CAM or CAM ligand conjugated to it may haveincreased ability to physically associate with cells or ECM thatcomprise a compatible CAM or CAM ligand, as compared with control cells.This property may be useful, e.g., if cells are used in regenerativemedicine or if it is desired that the cells integrate into an organ ortissue. An appropriate CAM or CAM ligand may be selected based on thesite at which it is desired that the cells associate or integrate. Forexample, a CAM or CAM ligand known to bind to a CAM or CAM ligandpresent at the site may be selected.

Selectins are a family of heterophilic CAMs that bind fucosylatedcarbohydrates. Selectins are involved in a variety of processesincluding constitutive lymphocyte homing and in chronic and acuteinflammation. The three selectin family members are E-selectin(endothelial), L-selectin (leukocyte), and P-selectin (platelet).Selectin ligands include P-selectin glycoprotein ligand-1 (PSGL-1),which is a mucin-type glycoprotein expressed on white blood cells.Selectin ligands include sialyl Lewis X (SLex)(NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc) and sialyl Lewis A (sLe(a))tetrasaccharides and a variety of structurally similar carbohydratemoieties, typically in the context of more extensive bindingdeterminants. Among other things, interaction between selectin ligandspresent on endothelial cell surfaces and selectins on leukocyte cellsurfaces mediates rolling and attachment of leukocytes to vessel wallsand facilitates their extravasation into tissues, e.g., at sites ofinflammation. Selectin binding to sLe(x) and sLe(a) present on variouscancer cell types, e.g., colon, gastric, bladder, pancreatic, breast,and prostate carcinomas, is implicated in enhancing metastasis. In someembodiments a selectin or selectin ligand is conjugated to mammaliancells using sortase. In some embodiments a selectin ligand serves as atarget or targeting moiety. For example, a selectin ligand may beconjugated to mammalian cells, e.g., immune systems cells, usingsortase. The cells may be administered to a subject, e.g.,intravascularly. The selectin may target the mammalian cells to sites ofinflammation, where endothelial cells express the cognate selectinligand. In some embodiments, e.g., if the inflammation is unwanted,e.g., associated with autoimmune disease or causing excessive symptoms,tissue damage or cytokine release, mammalian cells that haveimmunosuppressive activity (e.g., Tregs or cells that have beenstimulated to express an immunosuppressive cytokine or otherimmunosuppressive molecule or modified to have an immunosuppressivecytokine or other immunosuppressive molecule at their surface) may betargeted to the site. In some embodiments, e.g., if the inflammation isin response to an infection or other condition in which it may bebeneficial to stimulate an immune response, mammalian cells that have ormay stimulate appropriate effector responses may be targeted to thesite. In some embodiments mammalian cells that have a therapeutic agentconjugated thereto may be targeted to the site. In some embodiments aselectin serves as a target or targeting moiety. For example, a selectinmay be conjugated to mammalian cells, e.g., immune systems cells, usingsortase. The selectin may target the mammalian cells to tumor cells thatexpress the cognate selectin ligand. The mammalian cells may be immunesystem cells that have anti-tumor activity and/or may have a therapeuticagent, e.g., a cytotoxic agent, at their surface.

The invention encompasses application of the inventive methods to any ofthe proteins described herein and any proteins known to those of skillin the art. Without limitation, sequences of certain proteins ofinterest are found in, e.g., U.S. Ser. Nos. 10/773,530; 11/531,531; U.S.Ser. Nos. 11/707,014; 11/429,276; 11/365,008, and/or in Table T and/orunder the NCBI accession numbers listed in Table T or encoded by genesidentified by Gene ID and/or NCBI RefSeq accession number in Table T,described by Official Symbol (assigned by the HUGO Gene NomenclatureCommittee in the case of human genes), and/or described by name orotherwise herein. It is noted that where multiple isoforms of aparticular protein exist, the dominant isoform, longest isoform, isoform1, or isoform having a particular biological activity of interest may beselected. In some embodiments a cell-bound isoform may be selected. Insome embodiments a secreted isoform may be selected.

In some embodiments, modified versions of any protein, wherein themodified version comprises (i) one or more nucleophilic residues such asglycine at the N-terminus (e.g., between 1 and 10 residues) and,optionally, a cleavage recognition sequence, e.g., a protease cleavagerecognition sequence that masks the nucleophilic residue(s); or (ii) asortase recognition motif at or near the C-terminus may be used in acomposition or method described herein, e.g., attachment of the modifiedversion to a mammalian cell. In some embodiments, the protein comprisesboth (i) and (ii). In some aspects, the present disclosure providesproteins comprising any protein described herein, e.g., any antibody,antibody fragment, antibody chain, antibody domain, scFv, VHH, affibody,adnectin, anticalin, cytokine, cytokine chain, Ig superfamily protein,pro-apoptotic domain, or antigen, or a biologically active fragment orvariant of any of the foregoing; and a sequence comprising a sortaserecognition motif. In some embodiments the sortase recognition motif islocated at or near a C-terminus of the protein.

One of skill in the art will be aware that certain proteins, e.g.,secreted eukaryotic (e.g., mammalian) proteins, often undergointracellular processing (e.g., cleavage of a secretion signal prior tosecretion and/or removal of other portion(s) that are not required forbiological activity), to generate a mature form. Such mature,biologically active versions of proteins are used in certain embodimentsof the invention.

TABLE TSelected Protein Sequences and Selected Gene IDs and Accession NumbersTissue plasminogen activator (1rtf) Chain A: TTCCGLRQY (SEQ ID NO: 5)Chain B:  IKGGLFADIASHPWQAAIFAKHHRRGGERFLCGGILISSCWILSAAHCFQQQQQEEEEERRRRRFFFFFPPPPPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSSSSSDDDDDSSSSSSSSSSRRRRRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTTTSSSQQQHLLNRTVTDNMLCAGDTTTRRRSSSNNNLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGGQQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: XX) Factor IX Chain A:VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVEETTGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNNNAAAAAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTTTNNNIIIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGGFFHEGGGRRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAAMMKGKYGIYTKVSRYVNWIKEKTKLT (SEQ ID NO: 6) Chain B:MTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSK (SEQ ID NO: 7) GlucocerebrosidaseEFARPCIPKSFGYSSVVCVCNATYCDSFDPPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWHRQ (SEQ ID NO: 8)alpha galactosidase A LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTM (SEQ ID NO: 9)arylsulfatase-A (iduronidase, α-L-)RPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACCHCP (SEQ ID NO: 10)arylsulfatase B (N-acetylgalactos-amine-SRPPHLVFLLADDLGWNDVGFHGSRIRTPHLDALAAGGVLLDNY 4-sulfatase) (1fsu)YTQPLTPSRSQLLTGRYQIRTGLQHQIIWPCQPSCVPLDEKLLPQLLKEAGYTTHMVGKWHLGMYRKECLPTRRGFDTYFGYLLGSEDYYSHERCTLIDALNVTRCALDFRDGEEVATGYKNMYSTNIFTKRAIALITNHPPEKPLFLYLALQSVHEPLQVPEEYLKPYDFIQDKNRHHYAGMVSLMDEAVGNVTAALKSSGLWNNTVFIFSTDNGGQTLAGGNNWPLRGRKWSLWEGGVRGVGFVASPLLKQKGVKNRELIHISDWLPTLVKLARGHTNGTKPLDGFDVWKTISEGSPSPRIELLHNIDPNFVDSSPCSAFNTSVHAAIRHGNWKLLTGYPGCGYWFPPPSQYNVSEIPSSDPPTKTLWLFDIDRDPEERHDLSREYPHIVTKLLSRLQFYHKHSVPVYFPAQDPRCDPKATGVWGPWM (SEQ ID NO: 11)beta-hexosaminidase A (2gjx) LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAFQRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTPGHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQLESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPLAFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 12)Hexosaminidase A and B (2gjx) CHAIN A:LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAFQRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTPGHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQLESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPLAFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 13) Chain B:PALWPLPLSVKMTPNLLHLAPENFYISHSPNSTAGPSCTLLEEAFRRYHGYIFGTQVQQLLVSITLQSECDAFPNISSDESYTLLVKEPVAVLKANRVWGALRGLETFSQLVYQDSYGTFTINESTIIDSPRFSHRGILIDTSRHYLPVKIILKTLDAMAFNKFNVLHWHIVDDQSFPYQSITFPELSNKGSYSLSHVYTPNDVRMVIEYARLRGIRVLPEFDTPGHTLSWGKGQKDLLTPCYSDSFGPINPTLNTTYSFLTTFFKEISEVFPDQFIHLGGDEVEFKCWESNPKIQDFMRQKGFGTDFKKLESFYIQKVLDIIATINKGSIVWQEVFDDKAKLAPGTIVEVWKDSAYPEELSRVTASGFPVILSAPWYLDLISYGQDWRKYYKVEPLDFGGTQKQKQLFIGGEACLWGEYVDATNLTPRLWPRASAVGERLWSSKDVRDMDDAYDRLTRHRCRMVERGIAAQPLYAGYCN (SEQ ID NO: 14) Chain C:PALWPLPLSVKMTPNLLHLAPENFYISHSPNSTAGPSCTLLEEAFRRYHGYIFGTQVQQLLVSITLQSECDAFPNISSDESYTLLVKEPVAVLKANRVWGALRGLETFSQLVYQDSYGTFTINESTIIDSPRFSHRGILIDTSRHYLPVKIILKTLDAMAFNKFNVLHWHIVDDQSFPYQSITFPELSNKGSYSLSHVYTPNDVRMVIEYARLRGIRVLPEFDTPGHTLSWGKGQKDLLTPCYSLDSFGPINPTLNTTYSFLTTFFKEISEVFPDQFIHLGGDEVEFKCWESNPKIQDFMRQKGFGTDFKKLESFYIQKVLDIIATINKGSIVWQEVFDDKAKLAPGTIVEVWKDSAYPEELSRVTASGFPVILSAPWYLDLISYGQDWRKYYKVEPLDFGGTQKQKQLFIGGEACLWGEYVDATNLTPRLWPRASAVGERLWSSKDVRDMDDAYDRLTRHRCRMVERGIAAQPLYAGYCN (SEQ ID NO: 15) Chain D:LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAFQRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTPGHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQLESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPLAFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 16)phenylalanine hydroxylase (PAH) (1j8u)VPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVL (SEQ ID NO: 17) Cathepsin AAPDQDEIQRLPGLAKQPSFRQYSGYLKSSGSKHLHYWFVESQKDPENSPVVLWLNGGPGCSSLDGLLTEHGPFLVQPDGVTLEYNPYSWNLIANVLYLESPAGVGFSYSDDKFYATNDTEVAQSNFEALQDFFRLFPEYKNNKLFLTGESYAGIYIPTLAVLVMQDPSMNLQGLAVGNGLSSYEQNDNSLVYFAYYHGLLGNRLWSSLQTHCCSQNKCNFYDNKDLECVTNLQEVARIVGNSGLNIYNLYAPCAGGVPSHFRYEKDTVVVQDLGNIFTRLPLKRMWHQALLRSGDKVRMDPPCTNTTAASTYLNNPYVRKALNIPEQLPQWDMCNFLVNLQYRRLYRSMNSQYLKLLSSQKYQILLYNGDVDMACNFMGDEWFVDSLNQKMEVQRRPWLVKYGDSGEQIAGFVKEFSHIAFLTIKGAGHMVPTDKPLAAFTMFSRFLNKQPY (SEQ ID NO: 18) G-CSFLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMMPAFASAFQRRAGGVLVASHLQSFL EVSYRVLRHLA (SEQ ID NO: 19)GM-CSF EHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIP (SEQ ID NO: 20) Interferon alfa-2CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 21) Interferon beta-1MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 22) Interferon gamma-1bMQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIDELIQVMAELGANVSGEFVKEAENLKKYFNDNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAA (SEQ ID NO: 23) IL-2 (1M47)STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQNFHLRPRDLISNINVIVLELKGFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 24) IL-1 (2nvh)APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVS (SEQ ID NO: 25) TNF-alpha (4tsv)DKPVAHVVANPQAEGQLQWSNRRANALLANGVELRDNQLVVPIEGLFLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 26) TNF-beta (lymphotoxin) (1tnr)KPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSP STVFFGAFAL (SEQ ID NO: 27)Erythropoietin APPRLICDSRVLERYLLEAKEAEKITTGCAEHCSLNEKITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVKSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISNSDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO: 28) InsulinChain A: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 29)Chain B: FVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO: 30)Growth hormone (GH) (Somatotropin)FPTIPLSRLADNAWLRADRLNQLAFDTYQEFEEAYIPKEQIHSFW (1huw)WNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEALLKNYGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGF (SEQ ID NO: 31)Follicle-stimulating hormone (FSH)CHHRICHCSNRVFLCQESKVTEIPSDLPRNAIELRFVLTKLRVIQKGAFSGFGDLEKIEISQNDVLEVIEADVFSNLPKLHEIRIEKANNLLYINPEAFQNLPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINIHTIERNSFVGLSFESVILWLNKNGIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVFHGASGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLE (SEQ ID NO: 32) Leptin (1ax8)IQKVQDDTKTLIKTIVTRINDILDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPEASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID NO: 33)Insulin-like growth factor (orPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDE somatomedin) (1wqj)CCFRSCDLRRLEMYCAP (SEQ ID NO: 34) Adiponectin (1c28) Chain A:MYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMKDVKVSLFKKDKAVLFTYDQYQENVDQASGSVLLHLEVGDQVWLQVYYADNVNDSTFTGFLLYHDT (SEQ ID NO: 35) Chain B:MYRSAFSVGLPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMKDVKVSLFKKDKVLFTYDQYQEKVDQASGSVLLHLEVGDQVWLQVYDSTFTGFLLYHD (SEQ ID NO: 36) Chain C:MYRSAFSVGLETRVTVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVDVKVSLFKKDKAVLFTQASGSVLLHLEVGDQVWLQNDSTFTGFLLYHD (SEQ ID NO: 37) Factor VIII (aka antihemophilic factor)Chain A: (2r7e) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYED (SEQ ID NO: 38) Chain B:RSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYSSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY (SEQ ID NO: 39) Human serum albumin (1ao6) Chain A:SEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AA (SEQ ID NO: 40) Chain B:SEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AA (SEQ ID NO: 42)Hemoglobin (1bz0) Chain A: VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLA SVSTVLTSKYR (SEQ ID NO: 43)Chain B: VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH (SEQ ID NO: 44) Trail (TNFSF10)Gene ID: 8743; NP_003801.1 (isoform 1); NP_003801.1 (isoform 2);NP_001177872.1 (isoform 3) E-selectin (SELE) Gene ID: 6401; NP_000441.2CD3 epsilon (CD3E) Gene ID: 916; NP_000724.1 CD3 zeta (CD247)Gene ID: 919; NP_932170.1 (isoform 1);  NP_000725.1 (isoform 2)CD3 delta (CD3D) Gene ID: 915; NP_000723.1 (isoform A); NP_001035741.1 (isoform B) CD3 gamma (CD3G) Gene ID: 917; NP_000064.1CD28 Gene ID: NP_006130.1 (isoform 1);  NP_001230006.1 (isoform 2);NP_001230007.1 (isoform 3) Programmed cell death 1 (PD-1)Gene ID: 5133; NP_005009.2 (PDCD1) PD-L1 (CD274)Gene ID: 29126NP_054862.1 (isoform a);  NP_001254635.1 (isoform b);PD-L2 (PDCD1LG2) Gene ID: 80380; NP_079515.2 CTLA-4Gene ID: 1493; NP_005205.2 (isoform CLTA4-TM); NP_001032720.1 (isoform CTLA4delTM) BCMA (TNFRSF17)Gene ID: 608; NP_001183.2 CD137L (TNFSF9) Gene ID: 8744; NP_003802.1

It will be appreciated that considerable structure/function informationis available regarding many of the afore-mentioned proteins, as well assequences from different mammalian species, that can be used to designvariants of the naturally occurring sequence that retain significantbiological activity (e.g., at least 25%, 75%, 90%, 95%, 98%, 99%, ormore of the activity of the naturally occurring protein, or greateractivity than the naturally occurring protein). For example, crystalstructures or NMR structures of a number of proteins, in some instancesin a complex with the corresponding receptor, are available. It will beunderstood that a naturally occurring sequence can be extended, e.g., ator near its C-terminus, e.g., with a flexible peptide spacer (e.g., anyof the polypeptide linkers mentioned herein), which may allow thepolypeptide more freedom to fold and/or interact or associate with freeor cell-bound molecules or structures in the extracellular environmentafter it is conjugated to mammalian cells than would otherwise be thecase. In addition, it will be understood that, if the naturallyoccurring N- and C-termini are not located in close proximity to eachother in the native structure, a naturally occurring sequence can beextended at the N- and/or C-termini, e.g., with a flexible peptidespacer so that the termini can come into close proximity, which may bedesirable, for example, if the polypeptide is to be circularized.

In some embodiments, a protein has been tested in one or more humanclinical trials and, in some embodiments, demonstrated acceptable safetyin at least a Phase I trial. In some embodiments, a protein is approvedby the US Food & Drug Administration (or an equivalent regulatoryauthority such as the European Medicines Evaluation Agency) for use intreating a disease or disorder in humans. In some embodiments a proteinis a PEGylated version of the protein.

In some embodiments an agent conjugated to a living mammalian cell usingsortase comprises an antigen or a binding moiety that binds to anantigen. In some embodiments an antigen is any molecule or complexcomprising at least one epitope recognized by a B cell, e.g., amammalian or avian B cell and/or by a T cell, e.g., a mammalian or avianT cell. An antigen may comprise a polypeptide, a polysaccharide, acarbohydrate, a lipid, a nucleic acid, or combination thereof. In someembodiments an antigen comprises a protein, e.g., a polypeptide encodedor expressed by an organism. A polypeptide antigen may comprise orconsist of a full length polypeptide or a portion thereof, such as apeptide at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acidslong. In some embodiments an antigen comprises a lipid or glycolipidsuch as α-galactosylceramide (α-GalCer), which is recognized by iNKTcells. An antigen may be naturally occurring or synthetic. In someembodiments, an antigen is naturally produced by and/or is geneticallyencoded by a pathogen, an infected cell, or a neoplastic cell (e.g., acancer cell). In some embodiments, an antigen is an autoantigen (“selfantigen”), or an agent that has the capacity to initiate or enhance anautoimmune response. In some embodiments, an antigen is agraft-associated antigen. In some embodiments, an antigen is produced orgenetically encoded by a virus, bacteria, fungus, or parasite which, insome embodiments, is a pathogenic agent. In some embodiments, an agent(e.g., virus, bacterium, fungus, parasite) infects and, in someembodiments, causes disease in, at least one mammalian or avian species,e.g., human, non-human primate, bovine, ovine, equine, caprine, and/orporcine species. In some embodiments, a pathogen is intracellular duringat least part of its life cycle. In some embodiments, a pathogen isextracellular. In some embodiments an antigen comprises a molecule thatis produced by an infected cell as a result of infection by a pathogen.In some embodiments, an antigen is an envelope protein, capsid protein,secreted protein, structural protein, cell wall protein orpolysaccharide, capsule protein or polysaccharide, or enzyme. In someembodiments an antigen is a toxin, e.g., a bacterial toxin.

In some embodiments mammalian cells, e.g., hematopoietic cells, e.g.,immune system cells or red blood cells, that have an epitope, antigen orportion thereof conjugated thereto by sortase may be used as vaccinecomponents. As used herein, “vaccine” refers to a product or compositionthat may be administered to a subject to modulate the subject's immunesystem or immune response towards one or more entit(ies) of interest. Avaccine often contains an agent that resembles at least a portion of anentity against which an immune response is desired, e.g., adisease-causing microorganism, parasite, or toxin, or an agent thatresembles an entity against which an immune response is not desired,e.g., a self antigen or environmental allergen. In some embodiments anagent stimulates the body's immune system to recognize an entity asforeign, destroy it, and “remember” it (e.g., by inducing formation ofmemory T and/or B cells), so that the immune system can more easilyrecognize and destroy such an entity that it subsequently encounters. Insome embodiments an agent stimulates the body's immune system torecognize an entity as “self” or not to recognize the entity, so thatthe immune system does not mount a response against the entity. Forexample, it may be desirable to inhibit an immune response towards selfantigens or graft-associated antigens. A vaccine may be prophylactic(e.g., to prevent or reduce the severity of a future infection by apathogen or exposure to an allergen), or therapeutic (e.g., vaccinesagainst cancer or to treat autoimmune disease or an existing allergy).In some embodiments a vaccine modulates the adaptive immune system or acomponent thereof. In some embodiments a vaccine is designed to modulatethe immune response towards a single antigen, microorganism, or otherentity. In some embodiments a vaccine is designed to modulate the immuneresponse towards two or more strains of the same microorganism orentity, two or more microorganisms or entities, or two or more distinctantigens of a tumor, graft, or self cell or structure. Sortagged cellsused as a vaccine or vaccine component may be used to deliver anepitope, antigen or portion thereof to a subject in order to modulate animmune response of the subject towards an entity that comprises theepitope or antigen. In some embodiments an antigen conjugated to animalcells using sortase may be any antigen used in a conventional vaccineknown in the art.

One of ordinary skill in the art will be aware of numerous microbes(e.g., viruses, bacteria, fungi, protozoa) and multicellular parasitesfrom which antigens or epitopes may be derived, e.g., microbes andparasites capable of causing disease in mammals. In some embodiments anantigen is a surface protein or polysaccharide of, e.g., a viral capsid,envelope, or coat, or bacterial, fungal, protozoal, or parasite cell. Insome embodiments an antigen is a toxin, e.g., a toxin produced by abacterium. A toxin may be provided in an inactivated form, e.g., as atoxoid. An antigen or epitope may be modified, e.g., by chemicaltreatment (e.g., formaldehyde) or physical treatment (e.g., heat) and/orby conjugation with a second agent. It will be understood that anantigen, e.g., a protein, “derived from” a particular microbe orparasite can be produced using any suitable method, e.g., usingrecombinant DNA technology in yeast, bacteria, or cell cultures. In someembodiments a variant antigen may be used. For example, a nativesequence may be modified to render it more immunogenic. In someembodiments an antigen or epitope is sufficiently similar to a naturallyoccurring antigen or epitope such that it binds with at least about 10%,20%, 30%, least 50%, 60%, 70%, 80%, 90%, 95%, or the same affinity to anantigen receptor or antibody that binds to the naturally occurringantigen or epitope. In some embodiments an antigen or epitope issufficiently similar to a naturally occurring antigen or epitope toelicit a desired response.

Exemplary viruses include, e.g., Retroviridae (e.g., lentiviruses suchas human immunodeficiency viruses, such as HIV-I); Caliciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses, hepatitis C virus);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. Ebola viruses);Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus,respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);Bunyaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae(erg., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B or C virus); Parvoviridae (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae;Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zostervirus, cytomegalovirus (CMV), EBV, KSV); Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses).

Bacteria include, e.g., gram positive, gram negative, and acid-fastbacteria. Bacteria may be cocci, rod-shaped, spirochetes. Exemplarybacteria include, e.g., Helicobacter pylori, Borellia (e.g., B.burgdorferi, B. afzelii, B. garinii), Legionella pneumophilia,Mycobacteria (e.g., M. tuberculosis, M. avium, M, intracellulare, M.kansasii, M. gordonae), Staphylococcus (e.g., Staphylococcus aureus),Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, Campylobacter sp., Enterococcus sp., Chlamydiasp., Haemophilus influenzae, Bordetella (e.g., B. pertussis, B.parapertussis, B. bronchiseptica), Bacillus anthracis, Corynebacteriumdiphtheriae, Erysipelothrix rhusiopathiae, Clostridia (e.g., Clostridiumperfringens, Clostridium tetani, Clostridium difficile), Enterobacteraerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroidessp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidum, Treponema pertenue, Leptospira, Actinomyces israelii andFrancisella tularensis, E. coli (e.g., pathogenic E. coli).

In some embodiments a fungus is a member of the phylum Ascomycota,Basidiomycota, Chytridiomycota, Glomeromycota, or Zygomycota. The fungusmay be a member of a genus selected from the group consisting ofAspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus,Epidermophytum, Exserohilum, Fusarium, Histoplasma, Malassezia,Microsporum, Mucor, Paracoccidioides, Penicillium, Pichia, Pneumocystis,Pseudallescheria, Rhizopus, Rhodotorula, Scedosporium, Schizophyllum,Sporothrix, Stachybotrys, Saccharomyces, Trichophyton, Trichosporon,Bipolaris, Exserohilum, Curvularia, Alternaria, or Cladophialophora.Exemplary fungi include, e.g., Aspergillus, such as Aspergillus flavus,Aspergillus fumigatus, Aspergillus niger, Aspergillus clavatus,Blastomyces, such as Blastomyces dermatitidis, Candida, such as Candidaalbicans, Candida glabrata, Candida guilliermondii, Candida krusei,Candida parapsilosis, Candida tropicalis, Coccidioides, such asCoccidioides immitis, Cryptococcus, such as Cryptococcus neoformans,Epidermophyton, Fusarium, Histoplasma, such as Histoplasma capsulatum,Malassezia, such as Malassezia furfur, Microsporum, Mucor,Paracoccidioides, such as Paracoccidioides brasiliensis, Penicillium,such as Penicillium marneffei, Pichia, such as Pichia anomala, Pichiaguilliermondii, Pneumocystis, such as Pneumocystis carinii,Pseudallescheria, such as Pseudallescheria boydii, Rhizopus, such asRhizopus oryzae, Rhodotorula, such as Rhodotorula rubra, Scedosporium,such as Scedosporium apiospermum, Schizophyllum, such as Schizophyllumcommune, Sporothrix, such as Sporothrix schenckii, Trichophyton, such asTrichophyton mentagrophytes, Trichophyton rubrum, Trichophytonverrucosum, Trichophyton violaceutn, Trichosporon, such as Trichosporonasahii, Trichosporon cutaneum, Trichosporon inkin, and Trichosporonmucoides. In some embodiments a fungus is Coccidioides immitis,Coccidioides posadasii. Cryptococcus neoformans, C. gattii, C. albidus,C. laurentii, C. uniguttulas, E. floccosum, Fusarium graminearum,Fusarium oxysporum fsp. cubense, a member of the Fusarium solanicomplex, Fusarium oxysporum, Fusarium verticillioides, Fusariumproliferatum, Malassezia furfur, Mucor circinelloides, Paracoccidioidesbrasiliensis, Penicillium marneffei, Pichia anomala, Pichiaguilliermondi, Pneumocystis carinii, Pneumocystis jirovecii,Pseudallescheria boydii, Rhizopus oryzae, Rhodotorula rubra,Scedosporium apiospermum, Schizophyllum commune, Sporothrix schenckii,Trichophyton mentagrophytes, Trichophyton rubrum, Trichophytonverrucosum, Trichophyton tonsurans, or Trichophyton violaceum,Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin,Trichosporon mucoides, Exserohilum rostratum E. meginnisii, or E.longirostratum.

In some embodiments a parasite is a protozoan. In some embodiments theparasite belongs to the phylum Apicomplexa. Exemplary parasites include,e.g., parasites of the genus Plasmodium (Plasmodium falciparum,Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri,Plasmodium malariae, or Plasmodium knowlesi), Trypanosoma, Toxoplasma(e.g., Toxoplasma gondii), Leishmania (e.g., Leishmania major),Isospora, Schistosoma, or Cryptosporidium. In some embodiments a memberof the genus Cryptosporidium is C. parvum, C. hominis, C. canis, C.felis, C. meleagridis, or C. muris. In some embodiments a member of thegenus Isospora is Isospora belli. In some embodiments a member of thegenus Babesia is Babesia microti or Babesia divergens.

In some embodiments a protozoan is a member of a genus of amoebae. Insome embodiments a protozoan is a member of the genus Entamoeba. In someembodiments a member of the genus Entamoeba is Entamoeba histolytica,Entamoeba dispar, or Entamoeba moshkovskii. In some embodiments aprotozoan is a member of the genus Naegleria, e.g., Naegleria fowleri.In some embodiments a protozoan is a member of the genus Balamuthia,e.g., Balamuthia mandriallaris. In some embodiments a protozoan is amember of the genus Acanthameba. In some embodiments a member of thegenus Giardia is Giardia lamblia. In certain embodiments a protozoon isa member of genus Sarcocystis, e.g., Sarcocystis bovohominis,Sarcocystis suihominis or Sarcocystis bovicanis. In certain embodiments,a protozoon is a member of genus Cyclospora, e.g., Cyclosporacayetanensis. In certain embodiments, a protozoan is a member of genusNeospora, e.g., Neospora caninum. In certain embodiments, a protozoan isa member of genus Theileria e.g., Theileria parva. In certainembodiments, a protozoan is a member of genus Trichomonas, e.g.,Trichomonas vaginalis. In some embodiments a protozoan is akinetoplastid. In some embodiments a kinetoplastid is a trypanosomatid,e.g., a member of the genus Leishmania, e.g., L. donovani, L, major, L,tropica, or L. braziliensis, or a member of the genus Trypanosoma, e.g.,T, brucii, T. cruzii, T. congolense, or T. equiperdum.

In some embodiments a parasite resides extracellularly during at leastpart of its life cycle. Examples include nematodes, trematodes (flukes),and cestodes. In some embodiments an antigen may be from a nematode suchas Ascaris, Enterobius, Thichuris, and/or cestodes such as Taenia,Hymenolepis, and Echinococcus, a cestode such as Taenia, Hymenolepis,Echinococcus, or Fasciola, a trematode such as Schistosoma. In someembodiments an antigen is from Trichinella, Diphyllobothrium,Clonorchis, Paragonimus, Ancylostoma, Necator, Strongyloides,Wuchereria, Onchocerca, or Dracunculus. In some embodiments an antigenis from an intestinal helminth. In various embodiments an antigen canoriginate from any component of the parasite or can be derived fromparasites at any stage of their life cycle of the parasite, e.g., anystage that occurs within an infected organism such as a mammalian oravian organism. In some embodiments an antigen is derived from eggs ofthe parasite, cysts, or substances secreted by the parasite.

A graft-associated antigen may be any antigen expressed by or present ina transplanted tissue, organ, or cells. In some embodiments agraft-associated antigen is at least in part exposed at the surface oftransplanted cells. For example, a graft-associated antigen may be acell-surface protein expressed by transplanted cells. In someembodiments a graft-associated antigen is present in transplantedtissue, organ, or cells, but is absent or substantially absent (e.g.,not detectable using standard detection methods) in a subject(recipient) that receives the transplanted tissue, organ, or cells. Insome embodiments a graft-associated antigen is expressed by transplantedcells but is not expressed at a detectable level by receipient cells ofthe same cell type as the transplanted cells or, in some embodiments. Insome embodiments a graft is from a donor of a different species to therecipient (i.e., the graft is a xenograft), in which case many proteinsexpressed by the transplanted cells may be graft-associated antigens. Insome embodiments a graft-associated antigen is a polypeptide that ispolymorphic within the particular species to which the subject belongs.For example, human leukocyte antigens (HLA) antigens, e.g., majorhistocompatibility antigens class I (MHC I) and class II (MHCII) arehighly polymorphic.

In some embodiments an antigen is a tumor antigen (TA). In general, atumor antigen can be any antigenic substance produced by cells in atumor, e.g., tumor cells or in some embodiments tumor stromal cells(e.g., tumor-associated cells such as cancer-associated fibroblasts ortumor-associated vasculature). In many embodiments, a TA is a molecule(or portion thereof) that is differentially expressed by tumor cells ascompared with non-tumor cells. A TA may be expressed by a subset oftumors of a particular type and/or by a subset of cells in a tumor. A TAmay at least in part exposed at the cell surface of tumor cells or tumorstromal cells. In some embodiments a TA comprises an abnormally modifiedprotein, lipid, glycoprotein, or glycolipid. Tumor antigens may include,e.g., proteins that are normally produced in very small quantities andare expressed in larger quantities by tumor cells, proteins that arenormally produced only in certain stages of development, proteins whosestructure (e.g., sequence or post-translational modification(s)) ismodified due to mutation in tumor cells, or normal proteins that are(under normal conditions) sequestered from the immune system. In someembodiments, a TA is an expression product of a mutated gene, e.g., anoncogene or mutated tumor suppressor gene, an overexpressed oraberrantly expressed cellular protein, an antigen encoded by anoncogenic virus (e.g., HBV; HCV; herpesvirus family members such as EBV,KSV; papilloma virus, etc.), or an oncofetal antigen. Oncofetal antigensare normally produced in the early stages of embryonic development andlargely or completely disappear by the time the immune system is fullydeveloped. Examples are alphafetoprotein (AFP, found, e.g., in germ celltumors and hepatocellular carcinoma) and carcinoembryonic antigen (CEA,found, e.g., in bowel cancers and occasionally lung or breast cancer).Tyrosinase is an example of a protein normally produced in very lowquantities but whose production is greatly increased in certain tumorcells (e.g., melanoma cells). Other exemplary TAs include, e.g., CA-125(found, e.g., in ovarian cancer); MUC-1 (found, e.g., in breast cancer,ovarian cancer, and others); HER-2/neu (found, e.g., in breast cancer);melanoma-associated antigen (MAGE; found, e.g., in malignant melanoma);prostatic acid phosphatase (PAP, found in prostate cancer), Wilms' tumor1 protein (WT1, a transcription factor overexpressed in malignantmesothelioma, leukemias, and other solid tumors); CO17-1A (found, e.g.,in colon cancer), GD2 (a disialoganglioside expressed on tumors ofneuroectodermal origin, including human neuroblastoma and melanoma),epithelial cell adhesion molecule (Epcam; epithelial tumors). In someembodiments a TA is a cancer/testis (CT) antigen. CT antigens are afamily of proteins that are frequently expressed in a large variety ofmalignancies but are generally absent from healthy tissue, except forthe testis and placenta. CT antigens include NY-ESO-1 and LAGE-1. Insome embodiments a tumor antigen comprises human telomerase reversetranscriptase (hTERT). hTERT is a protein of 1132 amino acid residuesand is broadly expressed in cancers but exhibits little or no expressionin most normal somatic cells. In some embodiments a TA is an NKG2Dligand such as MICA, MICB, or ULBP1-6.

In some embodiments an antigen may be a protein that is found on normalB cells and plasma cells such as CD19 or CD20. These proteins may beuseful as a target in subjects with hematologic malignancies involvingsuch cells, such as various B cell malignancies, e.g., B cell lymphomas,acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),hairy cell leukemias (HCL), acute myelogenous leukemia (AML), andmultiple myeloma (MM). Other useful protein targets in hematologicmalignancies include CD22 in, e.g., HCL and ALL (e.g., B-ALL), CD30 in,e.g., Hodgkins lymphoma and anaplastic large cell lymphoma, CD37 in,e.g., CLL, and CD38 in, e.g., MM.

In some embodiments a tumor antigen is mesothelin (Gene ID: 10232 (MSLN;the gene encodes a precursor protein that is cleaved into two products,megakaryocyte potentiating factor and mesothelin); NP_001170826.1(isoform 1); NP_037536.2 (isoform 2). Mesothelin is aglycosylphosphatidylinositol (GPI) anchored cell surface protein that ishighly expressed in a variety of cancer such as mesothelioma, ovariancancer, pancreatic cancer, and is also expressed in lung adenocarcinoma,uterine serous carcinoma, cholangiocarcinoma, squamous cell carcinoma,and acute myeloid leukemia (Tang, Z, et al., Anticancer Agents Med Chem.2013 Feb. 1; 13(2): 276-280, and references therein). Mesothelin canbind to MUC16 (also known as CA-125), to mediate heterotypic celladhesion. A variety of agents that bind to mesothelin are known in theart. Such agents, or others, may be used as binding moieties ortargeting moieties. For example, MORAb-009 (amatuximab), is a chimericmonoclonal antibody containing a single chain murine variable region(scFv) (murine anti-mesothelin scFv SS1) and human IgGγ1 and k constantregions. A human mAb, m912 that specifically binds to cell surfaceassociated mesothelin was isolated from a human Fab library (Feng Y, etal., Mol. Cancer Ther. 2009; 8:1113-1118). A high-affinity human mAbnamed HN1 was identified based on a scFv isolated by phage displaytechnology (Ho M, et al., Int. J. Cancer. 2011; 128:2020-2030). In someembodiments mesothelin or a fragment or variant thereof that binds toCA-125 may be used as a binding moiety, e.g., to target cells to tumorcells that expess CA-125. In some embodiments the fragment of mesothelincomprises at least amino acids 296-359, consisting of 64 amino acids atthe N-terminal of cell surface mesothelin.

In some embodiments a tumor antigen is a glypican, e.g., glypican 3(GPC3). Glypicans are GPI-anchored proteins expressed by a variety ofdifferent cell types. Humans have six glypican proteins; GPC1-GPC6.Glypican 3 (GPC3) is expressed at high levels in certain tumors ofvarious types, including hepatocellular carcinoma, colorectal cancer,ovarian clear cell carcinoma (CCC), and melanoma. Glypican 1 (GPC1) isexpressed at high levels in a number of tumor types, including certainbreast cancers and pancreatic cancer (e.g., pancreatic ductaladenocarcinoma). A variety of agents that bind to glypicans are known inthe art. Such agents, or others, may be used as targeting moieties. Forexample. In some embodiments a subject with a tumor that has increasedGPC3 expression has an elevated level of a soluble GPC3 fragment intheir blood as compared with a normal level, as a result of cleavage ofcell surface GPC3. In some embodiments the binding moiety binds to anepitope that comprises a portion of the extracellular portion of theC-terminal domain of GPC3 (e.g., within the C-terminal ˜30 kD up toabout amino acid 560) so as to bind to GPC3 molecules that remainattached to the cell surface. A variety of antibodies that bind to GPC3are known in the art. Examples include monoclonal antibodies 1G12(Capurro M, et al. Gastroenterology. 2003; 125:89-97) and YP6, YP7, YP8,YP9 and YP9.1 (Phung, Y., et al., MAbs. 2012 Sep. 1; 4(5): 592-599) Ahumanized and stabilized version of the murine anti-GCP3 monoclonalantibody GC33 has been described (Nakano, K., et al, Anticancer Drugs.2010; 21:907-16). HN3 is a human heavy-chain variable domain antibodywith high affinity (Kd=0.6 nM) for cell-surface-associated GPC3molecules that recognizes a conformational epitope that requires boththe amino and carboxy terminal domains of GPC3 (Feng, M., et al., ProcNatl Acad Sci USA. 2013; 110(12):E1083-91).

In some embodiments an antigen is chondroitin sulfate proteoglycan-4(CSPG4). CSPG4 is highly expressed in melanoma, breast cancer (includingtriple negative breast cancer), head and neck squamous cell carcinoma,mesothelioma, glioblastoma, clear cell renal carcinoma, and sarcomas(Wang X, et al. Curr Mol Med. 2010 June; 10(4):419-29). A CSPG4-specificfully human single-chain antibody termed scFv-FcC21 has been described(Wang, X., et al., Cancer Res. (2011), 71(24):7410-22). scFv-FcC21 orits antigen-binding domain may be used to target cells to tumors.

In some embodiments an antigen may be a signaling lymphocyte activationmolecule (SLAM) family receptor, such as SLAM or SLAMf7 (also known asCS1 and CD319).

In some embodiments an antigen is B cell maturation factor (BCMA), alsoknown as B cell maturation antigen (BCMA, also known as CD269 andTNFRSF17). BCMA is a tumor necrosis family receptor (TNFR) member thatis expressed in cells of the B cell lineage, such as terminallydifferentiated B cells and plasma cells. BCMA delivers pro-survival cellsignals upon binding of its ligands, B cell activator of the TNF family(BAFF) and a proliferation inducing ligand (APRIL). Among other things,BCMA has functional activity in mediating the survival of B lineagecells such as plasma cells that maintain long-term humoral immunity. Theexpression of BCMA has also been linked to a number of cancers,autoimmune disorders, and infectious diseases. In some embodiments,cells are conjugated with a binding moiety that binds to BCMA. In someembodiments, cells conjugated with a binding moiety that binds to BCMAare used to deplete or inhibit a biological activity of cells thatexpress BCMA. An exemplary binding moiety is SG1, an antibody that bindsto BCMA and inhibits its activity (Ryan, M C, Mol Cancer Ther. 2007;6(11):3009-18). In some embodiments, cells that are conjugated with amoiety that binds to BCMA are cytotoxic immune cells, e.g., cytotoxic Tcells or NK cells and/or may have a cytotoxic moiety, e.g., apro-apoptotic moiety such as TRAIL, attached thereto. The moiety thatbinds to BCMA, the cytotoxic moiety, or both, may be attached to thecell, e.g., to a non-genetically engineered endogenous polypeptideexpressed by the cell, using sortase. In some embodiments the cells thatexpress BCMA are abnormally reactive and/or autoantibody-secretingplasma cells and/or B cells. Depletion or inhibition of such cells maybe useful in the treatment of a wide variety of autoimmune diseases,such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA),Sjogren's syndrome, or other autoimmune diseases. In some embodiments,cells conjugated with a moiety that binds to BCMA are used to depletecancer cells that express BCMA. BCMA is expressed on a number ofcancers, including a variety of hematologic malignancies such asHodgkin's and non-Hodgkin's lymphomas and multiple myeloma, and itsbiological relevance in maintaining the viability and proliferation ofvarious malignant cells has been demonstrated (see, e.g., Chiu, A, etal., Blood. 2007 Jan. 15; 109(2):729-39). BCMA expression has also beenfound on a variety of other tumor types such as glioblastoma, leukemia,Waldenstrom macroglobulinemia, and glioblastomas.

In some embodiments, an antigen is CD19. In some embodiments, cells areconjugated with a moiety that binds to CD19. In some embodiments, thebinding moiety comprises an antibody that binds to CD19. Examples ofsuch antibodies include XmAb5603 or XmAb5574 (Xenocor, Inc., Monrovia,Calif. and Morphosys, AG, Martinsried, Germany), which are IgG1,humanized MAbs (Horton H M, et al, Cancer Res 2008; 68(19):8049-8057).In some embodiments the murine monoclonal antibody FMC63 or a humanizedversion thereof is used as a binding moiety that binds to CD19. Incertain embodiments anti-CD19 monoclonal antibodies described in U.S.Pat. Pub. No. 20110104150 may be used.

In some embodiments a TA is expressed by tumor-associated stromal cells(e.g., tumor-associated fibroblasts or tumor-associated macrophages) orby tumor-associated vasculature. In some embodiments a TA is a componentof the modified subendothelial tumor extracellular matrix. Suchcomponent(s) may be secreted by tumor cells or tumor-associated cells.Examples include certain splice isoforms of fibronectin or oftenascin-C. Fibronectin is a large glycoprotein found in theextracellular matrix of mammalian tissues and plasma. Under tissueremodeling conditions, alternative splicing can lead to the insertion ofEDB, an extra 91-amino-acid type III homology domain, into fibronectin.EDB is typically undetectable in healthy individuals, but in manyaggressive solid tumors EDB is highly expressed around tumorvasculature. L19 is an antibody that recognizes EDB with high affinityand has been shown to localize to tumor blood vessels in animal modelsand cancer patients. Tenascins are glycoproteins found in theextracellular matrix of vertebrates. Isoforms of tenascin can arise intumors through alternative splicing at sites of neo-angiogenesis. The Cdomain of tenascin is undetectable in normal adult tissue but stronglyexpressed in a perivascular pattern in brain and lung tumors. The F16antibody recognizes the extra-domain A1 of tenascin and has shownselective accumulation at tumors and sites of inflammation ininflammatory disorders in animals and humans (see List, T. and Neri, D.Clinical Pharmacology: Advances and Applications 2013:5 (Suppl 1) 29-45,and references therein, for discussion of these and other antibodiesthat bind to the same antigens or other antigens of interest).

In some embodiments, an antigen is a molecular component (eg, histonesor DNA) that may be released at sites of cell death, such as necroticareas in tumors. Binding moieties, e.g., antibodies, that bind to suchantigens may be used to target cells to a tumor. For example, humanNHS76 is a phage display derived human monoclonal antibody thatrecognizes nucleic acids exposed by necrotic tumor cells as well asmetastases (Sharifi J, et al. Hybrid Hybridomics. 2001;20(5-6):305-312).

In some embodiments an antigen comprises a peptide. In some embodimentsthe peptide is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 aminoacids long. In some embodiments the peptide is between 20 and 50 aminoacids long. In some embodiments the peptide is between 8 and 30, between15 and 25, between 20 and 30, between 25 and 35, or between 35 and 50amino acids long. Peptides may bind directly to MHC molecules expressedon cell surfaces, may be ingested and processed by APC and displayed onAPC cell surfaces in association with MHC molecules, and/or may bind topurified MHC proteins (e.g., MHC oligomers). In some embodiments apeptide contains at least one epitope capable of binding to anappropriate MHC class I protein and/or at least one epitope capable ofbinding to an appropriate MHC class II protein. In some embodiments apeptide comprises a CTL epitope (e.g., the peptide can be recognized byCTLs when bound to an appropriate MHC class I protein). In someembodiments a peptide comprises a Th epitope (e.g., the peptide can berecognized by Th cells when bound to an appropriate MHC class IIprotein). In some embodiments the sequence of a peptide comprises orconsists of the sequence of a portion of a longer polypeptide that isnaturally encoded by a pathogen or a neoplastic cell or is produced byan infected cell as a result of the infection. In some embodiments anantigen is an artificial polypeptide whose sequence comprises multipledistinct sequences from different distinct polypeptides. For example,sequence of peptides that would be found as portions of distinctantigens in nature may be combined to produce a composite antigencomprising epitopes originating from such distinct antigens. Forexample, an antigen may comprise a polypeptide represented as X1-X2 . .. -Xn, where X1, X2 . . . Xn represent peptides found in distinctproteins, and in which n may range, e.g., from 2 to 5, 10, 20, or more.It will be understood that X1, X2, etc., may be directly adjacent toeach other or joined by intervening linker(s). The resulting compositeantigen may be capable of stimulating an immune response to multipledistinct antigens, e.g., each of the distinct antigens. In someembodiments multiple epitopes, e.g., multiple immunodominant epitopes,are combined to generate a composite antigen. In some embodiments thesequence of an antigen comprises multiple distinct variants of apolypeptide, wherein such variants are found in different strains,serotypes, or subtypes of a pathogen. For example, an antigen maycomprise peptides or polysaccharides obtained from at least 2, 5, 10,20, or more strains, serotypes, or subtypes (e.g., clades) of apathogen. In some embodiments the sequence of an antigen comprisesmultiple distinct variants of a polypeptide, wherein such variants arefound in different pathogenic strains or different pathogenic speciesbelonging to a particular genus. In some embodiments at least some ofthe different polypeptides are naturally encoded by the same pathogen.In some embodiments the different polypeptides are naturally encoded bydifferent pathogens. In some embodiments the different pathogens areviruses, bacteria, fungi, or parasites. In some embodiments the sequenceof an antigen comprises multiple distinct sequences from differentdistinct tumor antigens. In some embodiments an antigen is any antigenknown or used in the art as a vaccine or vaccine component. In someembodiments an epitope or antigen is a synthetic compound whose sequenceor structure resembles that of a naturally occurring epitope antigen.For example, in some embodiments the sequence of a naturally occurringepitope or antigen may be altered by addition, deletion, or substitutionof one or more amino acids. In some embodiments an epitope or antigencomprises a portion at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%,99%, or more identical in sequence to at least a portion of a naturallyoccurring polypeptide, wherein the portion of the naturally occurringpolypeptide is at least 10; 20; 30; 40; 50; 100; 200; 500; 1,000; 2,000;3,000, or more amino acids long.

In some embodiments epitopes may be provided as a pool of peptides,which may be derived from one or more proteins. The protein(s) mayinclude one or more proteins that are known to be target(s) ofcell-mediated and/or humural immunity in at least some individuals. Insome embodiments a mixture of peptides may contain at least one epitopecapable of binding to MHC class I proteins and at least one epitopecapable of binding to MHC class II proteins. In some embodiments amixture of peptides may contain at least one CTL epitope and at leastone Th epitope. A peptide pool may comprise multiple epitopes that canbind to different MHC alleles. Peptides in the pool may bind to MHCalleles from individuals of diverse genetic backgrounds and may becapable of stimulating T and/or B cells from individuals of diversegenetic backgrounds. The peptides may be from 8 to 30 amino acids (aa)long, e.g., 9 to 15 aa long, 15 to 25 aa long. In some embodiments thepeptides may be generated by chemical and/or enzymatic partialhydrolysis of longer protein(s). In some embodiments the peptidescomprise a mixture of overlapping synthetic peptides. Overlappingsynthetic peptides typically represent sequential stretches of aminoacids, wherein a given peptide within the pool overlaps with neighboringpeptides by at least one aa up to n−1 aa, wherein n is the length of thepeptides. The first aa of a given peptide may be offset from the firstaa of its neighboring peptides by from 1 aa up to n−1, wherein n is thelength of the peptides. In some embodiments the offset is 2, 3, 4, or 5amino acids. For example, individual peptides may be 15 aa in length (15mer) and overlap with their neighboring peptides by 11 aa (offset=4).Starting at position 1 of a 30 amino acid polypeptide, such a peptidepool would contain peptides extending from aa 1-15, aa 5-20, aa 10-25,and aa 15-30. As another example, peptides may be 20 aa in length, with10 aa overlaps between sequential peptides (offset=10). Peptides in apool may be, but need not be, the same length. A peptide pool mayinclude some peptides that fall completely within the sequence of otherpeptides. The peptides may cover all or part of the full length of apolypeptide. In some embodiments a peptide pool comprises peptides thatcollectively encompass at least 50%, 60%, 70%, 80%, 90%, 95%, or all 9peptide sequences of a particular protein or proteins. The number ofdifferent peptides in a peptide pool may range from 2 up to about 300,up to about 500, up to about 1,000, or more. In some embodiments thenumber of different peptides is at least 10, 20, 30, 40, 50, e.g.,between 20 and 100, 100 and 200, 200 and 300, 300 and 400, or 400 and500. The peptides may be synthesized using standard solid phase peptidesynthesis methods.

In general, an antigen or epitope that originates from a particularsource may, in various embodiments, be isolated from such source or maybe produced using any appropriate means, e.g., using recombinant nucleicacid technology or chemical synthesis, or combinations thereof. Anantigen or epitope may be modified, e.g., by conjugation to anothermolecule or entity (e.g., an adjuvant), chemical or physicaldenaturation, etc. In certain embodiments an antigen or antigencomposition comprises or is derived at least in part from cells ortissues. For example, a tumor or tumor sample may be removed from asubject and used to isolate or identify one or more antigens present inthe tumor or tumor sample. Such antigens may be used, e.g., to stimulateimmune cells ex vivo, to generate or select binding agents (e.g.,antibodies) that bind to the antigen(s).

Antigens may be useful for a variety of purposes. It will be understoodthat epitopes derived from a particular antigen may be used for any suchpurpose in certain embodiments. For example, an antigen may be used toidentify, generate, test, or use an antibody or other agent that bindsto the antigen or may be conjugated to another entity, e.g., to apolypeptide or cell. Antigens may be used in vitro to load or stimulatecells of the immune system. For example, antigens may be contacted withAPCs to cause such APCs to take up, process, and display epitopes at thecell surface. The APCs may be administered to a subject or may be usedin vitro to stimulate cells of the adaptive immune system (e.g., naïve Tand/or B cells), which may subsequently be administered to a subject.

Pathogen-derived antigens may be useful in, e.g., identifying ordetecting pathogens or pathogen-infected cells (e.g., for purposes ofdiagnosis of an infection, for purposes of monitoring subjects who havereceived treatment for an infection e.g., to test for recurrence), forpurposes of targeting various agents (e.g., therapeutic agents) topathogens or pathogen-infected cells, and/or for purposes of modulating(e.g., directing or enhancing) an immune response towards pathogens orpathogen-infected cells. Tumor antigens may be useful in, e.g.,identifying or detecting tumor cells or tumor-associated cells (e.g.,for purposes of diagnosis, for purposes of monitoring subjects who havereceived treatment for a tumor, e.g., to test for recurrence), forpurposes of targeting, e.g., targeting therapeutic agents or cells, totumor cells or tumor-associated cells, and/or for purposes of modulating(e.g., directing or enhancing) an immune response towards tumor cells ortumor-associated cells.

Self antigens may be useful in, e.g., identifying or detectingantibodies or immune system cells that may contribute to an autoimmunedisease (e.g., for purposes of diagnosis of an autoimmune disease, forpurposes of monitoring subjects who have received treatment for anautoimmune disease, e.g., to test for recurrence), identifying ordetecting self cells or substances towards which an inappropriate, e.g.,harmful or potentially harmful, immune response is directed in anautoimmune disease (e.g., for purposes of diagnosis or treatmentselection), for purposes of targeting various agents (e.g., therapeuticagents) to self cells or substances towards which an inappropriate,e.g., harmful or potentially harmful, immune response is directed in anautoimmune disease, and/or for purposes of modulating (e.g., inhibiting)an immune response towards cells or substances towards which aninappropriate, e.g., harmful or potentially harmful, immune response isdirected in an autoimmune disease.

Graft-associated antigens may be useful in, e.g., identifying ordetecting antibodies or immune system cells that may contribute to graftrejection (e.g., for purposes of diagnosis of graft rejection, forpurposes of monitoring subjects who have received a graft), identifyingor detecting grafted cells towards which a harmful or potentiallyharmful immune response is directed in a subject who has received agraft (e.g., for purposes of diagnosis or treatment selection), forpurposes of targeting various agents (e.g., therapeutic agents) tografted cells towards which a harmful or potentially harmful immuneresponse is directed in in a subject who has received a graft, and/orfor purposes of modulating (e.g., inhibiting) an immune response towardsgrafted cells in a subject who has received a graft.

In some embodiments an antigen is a target for a targeting moiety thattargets an entity, e.g., a cell, detection agent, or therapeutic agent,to a pathogen, pathogen-infected cell, tumor cell, or tumor associatedcell. In some embodiments an antigen is a target for a targeting moietythat targets an entity, e.g., a cell, detection agent or therapeuticagent, towards a cell or substance comprising a self antigen or otherantigen towards which tolerance is desired, such as a graft-associatedantigen. In some embodiments an agent conjugated to a living mammaliancell using sortase comprises a moiety that binds to an antigen, such asan antibody or antibody fragment.

In some embodiments a protein comprises an antibody, antibody fragment,or antibody domain. In some embodiments a protein comprising anantibody, antibody fragment, or antibody domain is conjugated to livingmammalian cells using sortase. The cells may be used as a deliveryvehicle for the protein and/or the protein may serve as a targetingmoiety to target the cells to a target to which the antibody, antibodyfragment, or antibody domain binds. In some embodiments a protein is atherapeutic antibody. Exemplary therapeutic antibodies that are usefulin various embodiments provided herein include, but are not limited to,the following antibodies (target of the antibody is listed inparentheses together with exemplary non-limiting therapeuticindications): Abciximab (glycoprotein IIb/IIIa; cardiovascular disease),Adalimumab (TNF-α, various auto-immune disorders, e.g., rheumatoidarthritis), Alemtuzumab (CD52; chronic lymphocytic leukemia),Basiliximab (IL-2Rα receptor (CD25); transplant rejection), Bevacizumab(vascular endothelial growth factor A; various cancers, e.g., colorectalcancer, non-small cell lung cancer, glioblastoma, kidney cancer; wetage-related macular degeneration), Blinatumomab (anti-CD3/anti-CD19;various hematologic malignancies), Brentuximab (CD30; varioushematologic malignancies); Catumaxomab (CD3 and EpCAM; malignantascites), Cetuximab (EGF receptor, various cancers, e.g., colorectalcancer, head and neck cancer), Certolizumab (e.g., Certolizumab pegol)(TNF alpha; Crohn's disease, rheumatoid arthritis), Daclizumab (IL-2Rαreceptor (CD25); transplant rejection), Eculizumab (complement proteinC5; paroxysmal nocturnal hemoglobinuria), Efalizumab (CD11a; psoriasis),Elotuzumab (CD1 (also known as SLAMF7 and as CD319, multiple myeloma);Epratuzumab (CD22; Non-Hodgkin's lymphoma; lupus; ALL); Gemtuzumab(CD33; acute myelogenous leukemia (e.g., with calicheamicin)),Ibritumomab tiuxetan (CD20; Non-Hodgkin lymphoma (e.g., with yttrium-90or indium-111)), Infliximab (TNF alpha; various autoimmune disorders,e.g., rheumatoid arthritis), (Ipilimumab; CTLA-4, melanoma, prostatecancer), Milatuzumab (CD74; CD74-positive hematologic malignancies andsolid tumors); Muromonab-CD3 (T Cell CD3 receptor; transplantrejection), Natalizumab (alpha-4 (a4) integrin; multiple sclerosis,Crohn's disease), Nivolumab (PD-1; cancer, e.g., non-small-cell lungcancer, melanoma, and renal-cell cancer); Omalizumab (IgE;allergy-related asthma); Ofatumumuab (CD20; Non-Hodgkin lymphoma,chronic lymphocytic leukemia); Obinutuzumab (CD20; Non-Hodgkin lymphoma,chronic lymphocytic leukemia); Palivizumab (epitope of RSV F protein;Respiratory Syncytial Virus infection), Panitumumab (EGF receptor;cancer, e.g., colorectal cancer), Ranibizumab (vascular endothelialgrowth factor A; wet age-related macular degeneration), Rituximab (CD20;Non-Hodgkin lymphoma), Tositumomab (CD20; Non-Hodgkin lymphoma),Trastuzumab (ErbB2; breast cancer); Tremelimumab (CTLA-4, melanoma);Veltuzumab (CD20; Non-Hodgkin lymphoma, chronic lymphocytic leukemia),and any antigen-binding fragment thereof. In some embodiments anantibody or other binding agent binds to the same target as any of theafore-mentioned antibodies. In some embodiments an antibody or otherbinding agent competes with any of the afore-mentioned antibodies forbinding to its target. It will be understood that antigen bindingdomains of any of the afore-mentioned antibodies or others described maybe used. For example, Fab fragments or single chain variable fragments(scFv) may be used.

In some embodiments a binding moiety, e.g., an antibody, binds to anextracellular domain of a mammalian receptor. In some embodiments thereceptor is overexpressed in a tumor cell as compared with a normalcell, e.g., a normal cell of the same cell type, and/or has increasedactivity in a tumor cell as compared with a normal cell, e.g., a normalcell of the same cell type. In some embodiments the receptor is encodedby a gene that is mutated, is a fusion gene that results from achromosomal translocation, and/or is amplified in a tumor cell. In someembodiments the receptor is a protein kinase, e.g., a tyrosine kinase ora serine/threonine kinase. In some embodiments the receptor is anoncogenic protein kinase.

In some embodiments, a therapeutic monoclonal antibody and a secondagent useful for treating the same disease or comprising a targetingmoiety are conjugated to mammalian cells using sortase. In someembodiments, the second agent comprises a polypeptide, peptide, smallmolecule, or second antibody.

In some embodiments, a monoclonal antibody and a cytokine, e.g., aninterferon, e.g., interferon alpha, are conjugated to mammalian cellsusing sortase. Optionally, the monoclonal antibody and cytokine are bothuseful for treating the same disease.

In some embodiments one or more subunits (e.g., separate polypeptidechains) of a multisubunit protein (which term is used interchangeablywith multichain protein) is conjugated to mammalian cells using sortase.In some embodiments, a multisubunit protein is a receptor (e.g., a cellsurface receptor). In some embodiments, a multisubunit protein is anenzyme. In some embodiments, a multisubunit protein is a cytokine. Insome embodiments, a multisubunit protein is a channel or transporter. Insome embodiments at least one subunit of a multisubunit proteincomprises a sortase recognition motif, which may be used to conjugatethe subunit to mammalian cells or to conjugate a moiety to thepolypeptide. Various multisubunit polypeptides and methods of modifyingthem using sortase are described in WO/2011/133704. In some embodimentsa sortase recognition motif is located in a flexible loop, which may becleaved by a protease so as to position the sortase recognition motif ator near the C-terminus of a resulting cleavage product. In someembodiments a first subunit of a multisubunit protein is conjugated tomammalian cells. In some embodiments one of more additional subunits maysubsequently associate with the first subunit, e.g., to form a completemulti-subunit protein. In some embodiments such association may occur invitro. The one or more additional subunits may be added to a culturevessel or may be produced by cells in the vessel. In some embodimentsassociation occurs in vivo after administration of the cells to asubject. In some embodiments the one or more additional subunits may beproduced by the administered cells, by other cells in the body of thesubject, or may be administered to the subject. In some embodiments twoor more subunits, at least one of which comprises a sortase recognitionmotif, assemble to form a multi-subunit protein before conjugation tomammalian cells. In some embodiments two or more subunits are covalentlylinked to each other directly or via a linker before the protein isconjugated to mammalian cells. In some embodiments, such linkagefacilitates proper folding of the multi-subunit protein (e.g.,accelerates folding or increases proportion of correctly foldedfunctional proteins). In some embodiments a subunit of a multi-subunitprotein may be modified using sortase before the subunit or a differentsubunit of the protein is conjugated to mammalian cells. For example, alabel may be conjugated to a first subunit using sortase, and a secondsubunit may be conjugated to cells. The various conjugation and/orassociation steps may occur in any order in various embodiments.

In some embodiments a protein comprises a peptide that binds to atarget. In some embodiments the peptide is selected using a displaytechnology, e.g., phage display, yeast display, ribosome display,bacterial display, or directed evolution. In some embodiments thepeptide is selected from a peptide library. In some embodiments aprotein may comprise any of a variety of polypeptide scaffolds known inthe art including, e.g., those based on or incorporating one or moreprotein folds or domains from, e.g., protein Z, fibronectin, ankyrinrepeat proteins; cysteine-knot miniproteins, Armadillo repeat proteins,lipocalins, or stefin A. In some embodiments a protein comprises anaffibody, adnectin, DARPin, knottin, anticalins, or steffin. Theprotein, e.g., affibody, adnectin, DARPin, knottin, anticalins, orsteffin, may be designed or selected to bind to a target of interest.Such engineered binding proteins may in some embodiments have aspecificity and/or affinity comparable to or in some embodimentssuperior to that of typical antibodies. In some embodiments a peptidethat binds to a target is inserted into a polypeptide scaffold. See,e.g., Hoffmann, T., et al. Protein Eng Des Sel., 23(5):403-13, 2010, andreferences therein, for discussion of various proteins and polypeptidescaffolds. In some embodiments any such protein or scaffold is used,e.g., as a binding moiety, targeting moiety, immunomodulator, ortherapeutic agent.

In some embodiments a binding agent or moiety, e.g., an antibody, bindsto a target antigen, target entity, or binding partner with a K_(D) ofless than about 10⁻⁶ M, less than about 10⁻⁷ M, less than about 10⁻⁸ Mless than about 10⁻⁹ M, less than about 10⁻¹⁰ M, less than about 10⁻¹¹M,less than about 10⁻¹²M, or less than about 10⁻¹³M. In certainembodiments a binding agent or moiety binds to a target antigen ortarget entity or binding partner with a K_(D) of between about 10⁻⁶ Mand about 10⁻¹³M, e.g., between about 10⁻⁶ M and about 10⁻⁷M, betweenabout 10⁻⁷M and about 10⁻⁸M, between about 10⁻⁸M and about 10⁻⁹M,between about 10⁻⁹M and about 10⁻¹⁰ M, between about 10⁻¹⁰ M and about10⁻¹¹ M, or between about 10⁻¹¹ M and about 10⁻¹² M or between about10⁻¹² M and about 10⁻¹³ M. In some embodiments a binding interaction mayhave a K_(D) of between about 10⁻¹⁶M and about 10⁻¹²M.

In certain embodiments nucleic acids, e.g., short interfering RNAs,antisense oligonucleotides, or aptamers, may be conjugated to mammaliancells using sortase or used in vitro, e.g., to promote or inhibitexpansion, activation, or differentiation of cells. In some embodimentsnuclieic acid aptamers are of interest, e.g., as binding moieties,targeting moieties, immunomodulators, therapeutic agents, ligands. Anaptamer comprises an oligonucleotide that binds specifically and withhigh affinity to its target (e.g., a protein target). In someembodiments the oligonucleotide is single-stranded (although it may insome embodiments form regions of double-stranded secondary structurethrough intramolecular complementarity). An aptamer may be identifiedthrough a selection process using, e.g., systematic evolution of ligandsby exponential enrichment (SELEX), phage display, or various directedevolution techniques. See, e.g., Turek, C. and Gold, L., Science 249:505-10, 1990; Brody E N and Gold L J, Biotechnol. J, 74(1):5-13, 2000;L. Cerchia and V. de Franciscis, Trends Biotechnol., 28: 517-525, 2010;Keefe, A. Nat. Rev. Drug Discov. 9: 537-550, 2010. Aptamers can begenerated using DNA or RNA backbones, either of which may comprise anyof a variety of modifications such as substitution of ribonucleotideswith 2′-amino, 2′-fluoro, or 2′-O-alkyl nucleotides. Aptamers can begenerated against most targets and can inhibit the function of theproteins to which they bind or may act as agonists to activate areceptor to which they bind. Aptamers may be, e.g., about 25 to 80 ntlong and can be synthesized chemically.

In some embodiments small molecules may be used, e.g., as targetingmoieties, immunomodulators, detection agents, therapeutic agents, or asligands to activate or inhibit a receptor.

In some embodiments an agent to be conjugated to mammalian cellscomprises an anti-cancer agent (also termed a “chemotherapy drug”). Incertain embodiments cells are conjugated both with an anti-cancer agentand a targeting moiety, wherein the targeting moiety targets the cell toa cancer, which in some embodiments is a cancer of a type that istypically treated with the anti-cancer agent. In certain embodimentscells conjugated with an anti-cancer agent and/or with a targetingmoiety that targets the cells to a cancer are administered to a subjectwho is in need of treatment for cancer. Any anti-cancer agent may beused in various embodiments. In some embodiments an anti-cancer agent isa protein, e.g., a monoclonal antibody. In some embodiments ananti-cancer agent is an enzyme, e.g., asparaginase. Non-limitingexamples of chemotherapy drugs that may be used include, e.g.,alkylating and alkylating-like agents such as nitrogen mustards (e.g.,chlorambucil, chlormethine, cyclophosphamide, ifosfamide, andmelphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine,streptozocin); platinum agents (e.g., alkylating-like agents such ascarboplatin, cisplatin, oxaliplatin, BBR3464, satraplatin), busulfan,dacarbazine, procarbazine, temozolomide, thioTEPA, treosulfan, anduramustine; antimetabolites such as folic acids (e.g., aminopterin,methotrexate, pemetrexed, raltitrexed); purines such as cladribine,clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine;pyrimidines such as capecitabine, cytarabine, fluorouracil, floxuridine,gemcitabine; spindle poisons/mitotic inhibitors such as taxanes (e.g.,docetaxel, paclitaxel), vincas (e.g., vinblastine, vincristine,vindesine, and vinorelbine), epothilones; cytotoxic/anti-tumorantibiotics such anthracyclines (e.g., daunorubicin, doxorubicin,epirubicin, idarubicin, mitoxantrone, pixantrone, and valrubicin),compounds naturally produced by various species of Streptomyces (e.g.,actinomycin, bleomycin, mitomycin, plicamycin) and hydroxyurea;topoisomerase inhibitors such as camptotheca (e.g., camptothecin,topotecan, irinotecan) and podophyllums (e.g., etoposide, teniposide);monoclonal antibodies for cancer therapy such as anti-receptor tyrosinekinases (e.g., cetuximab, panitumumab, trastuzumab), anti-CD20 (e.g.,rituximab and tositumomab), and others for example alemtuzumab,aevacizumab, gemtuzumab; photosensitizers such as aminolevulinic acid,methyl aminolevulinate, porfimer sodium, and verteporfin; tyrosineand/or serine/threonine kinase inhibitors, e.g., inhibitors of Abl, Kit,insulin receptor family member(s), VEGF receptor family member(s), EGFreceptor family member(s), PDGF receptor family member(s), FGF receptorfamily member(s), mTOR, Raf kinase family, phosphatidyl inositol (PI)kinases such as PI3 kinase, PI kinase-like kinase family members, cyclindependent kinase (CDK) family members, Aurora kinase family members(e.g., kinase inhibitors that are on the market or have shown efficacyin at least one phase III trial in tumors, such as cediranib,crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib,nilotinib, sorafenib, sunitinib, vandetanib), growth factor receptorantagonists, and others such as retinoids (e.g., alitretinoin andtretinoin), altretamine, amsacrine, anagrelide, arsenic trioxide,asparaginase (e.g., pegasparagase), bexarotene, bortezomib, denileukindiftitox, estramustine, ixabepilone, masoprocol, mitotane, andtestolactone, Hsp90 inhibitors, proteasome inhibitors (e.g.,bortezomib), angiogenesis inhibitors, e.g., anti-vascular endothelialgrowth factor agents such as bevacizumab (Avastin) or VEGF receptorantagonists or soluble VEGF receptor domain (e.g., VEGF-Trap), matrixmetalloproteinase inhibitors, various pro-apoptotic agents (e.g.,apoptosis inducers), Ras inhibitors, anti-inflammatory agents, cancervaccines, or other immunomodulating therapies, RNAi agents targeted tooncogenes, etc. It will be understood that the preceding classificationis non-limiting. A number of anti-tumor agents have multiple activitiesor mechanisms of action and could be classified in multiple categoriesor classes or have additional mechanisms of action or targets.

In some embodiments an agent to be conjugated to mammalian cellscomprises an anti-microbial agent. As used herein, anti-microbial agentsinclude compounds that inhibit proliferation or activity of, weaken,destroy, or kill bacteria, viruses, fungi, parasites (e.g., protozoa,helminths (whether or not microscopic) compounds that inhibit invasionof cells by viruses, bacteria, or parasites; compounds that inhibit oneor more steps of a viral, bacterial, fungal, or parasite life cycle. Incertain embodiments cells are conjugated with an anti-microbial agentsuitable for use against a bacteria, virus, fungi, or parasite and witha targeting moiety, wherein the targeting moiety targets the cell to thebacteria, virus, fungi, or parasite or targets the cell to a cellinfected by the bacteria, virus, fungi, or parasite.

One of skill in the art will be aware of or can readily obtain thesequences of proteins described herein or other proteins of interest.Naturally occurring sequences, e.g., genomic, mRNA, and polypeptidesequences, from a wide variety of species, including human, are known inthe art and are available in publicly accessible databases such as thoseavailable at the National Center for Biotechnology Information(www.ncbi.nih.gov) or Universal Protein Resource (www.uniprot.org).Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt,UniProtKB/Trembl, and the like. In general, sequences, e.g., nucleicacid (e.g., mRNA) and polypeptide sequences, in the NCBI ReferenceSequence database may be used as reference sequences. It will beappreciated that multiple alleles of a gene may exist among individualsof the same species. For example, differences in one or more nucleotides(e.g., up to about 1%, 2%, 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies. Due to the degeneracy of the genetic code, such variationsoften do not alter the encoded amino acid sequence, although DNApolymorphisms that lead to changes in the sequence of the encodedproteins can exist. Examples of polymorphic variants can be found in,e.g., the Single Nucleotide Polymorphism Database (dbSNP), available atthe NCBI website at www.ncbi.nlm.nih.gov/projects/SNP/. (Sherry S T, etal. (2001). “dbSNP: the NCBI database of genetic variation”. NucleicAcids Res. 29 (1): 308-311; Kitts A, and Sherry S, (2009). The singlenucleotide polymorphism database (dbSNP) of nucleotide sequencevariation in The NCBI Handbook [Internet]. McEntyre J, Ostell J,editors. Bethesda (Md.): National Center for Biotechnology Information(US); 2002(www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch5).Multiple isoforms of certain proteins may exist, e.g., as a result ofalternative RNA splicing or editing. In general, where aspects of thisdisclosure pertain to a gene or gene product, embodiments pertaining toallelic variants or isoforms are encompassed unless indicated otherwise.Certain embodiments may be directed to particular sequence(s), e.g.,particular allele(s) or isoform(s). It will be understood that apolypeptide may be encoded by any of numerous different nucleic acidsequences due to the degeneracy of the genetic code. If a polypeptide isproduced recombinantly, a nucleic acid sequence encoding the polypeptidemay be selected or codon optimized for expression in a particularspecies, if desired. It should be understood that wherever reference ismade herein to a protein or polypeptide, e.g., a naturally occurringprotein or polypeptide, the invention provides embodiments in which avariant or fragment, e.g., a functional variant or fragment, may beused. (See discussion of variants and fragments above). For example, itwill be understood that an enzyme conjugated to mammalian cells forpurposes of supplementing or replacing an enzyme that is lacking orpresent in insufficient amounts need not be identical in sequence to anaturally occurring enzyme that is lacking or insufficient, so long asit provides the appropriate catalytic activity.

In some embodiments a protein or other moiety conjugated to mammaliancells is PEGylated. In some embodiments PEGylation is may beaccomplished using sortase, e.g., before the protein or other moiety isconjugated to mammalian cells. For example, a protein may be PEGylatedat its N-terminus and then conjugated to mammalian cells via itsC-terminus. In some embodiments sortase is used to conjugate a moietycomprising PEG to mammalian cells.

Sortagged cells, e.g., sortagged mammalian cells, described herein havea number of uses. Some of these uses are described herein, but theinvention is not limited to uses described herein. In some embodimentssortagged mammalian cells may be used in cell therapy. As used herein,the terms “cell therapy”, “cell-based therapy”, or “cellular therapy”are used interchangeably to refer to administration of eukaryotic cells,e.g., mammalian cells, to a subject for therapeutic purposes. In someembodiments cell therapy comprises cell-based immunotherapy, whichrefers to administration of cells to a subject in order to modulate oraugment the subject's immune system or immune response for therapeuticpurposes. Cell-based immunotherapy encompasses administering immunesystem cells to a subject, wherein the administered cells or theirdescendants, through their own effector mechanisms and/or throughinteractions with cells or substances of the subject's immune system,may provide a therapeutic benefit to the subject. It will be understoodthat where the present disclosure refers to the effects of administeredcells, such effects encompass the effects of the administered cells andtheir descendants that are generated in vivo. In some embodiments celltherapy is administered for treatment of cancer, infections, autoimmunediseases, or enzyme deficiencies. In certain embodiments administeredcells may originate from the individual to whom they are administered(autologous), may originate from different genetically identicalindividual(s) of the same species (isogeneic), may originate fromdifferent non-genetically identical individual(s) of the same species(allogeneic), or may originate from individual(s) of a differentspecies. In certain embodiments allogeneic cells may originate from anindividual who is immunocompatible with the subject to whom the cellsare administered.

In some embodiments living mammalian cells modified by sortase-mediatedconjugation of an agent thereto are used as delivery vehicles for theagent. For example, living mammalian cells that have a proteinconjugated to their surface may serve as delivery vehicles for theprotein. Such cells may be administered to a subject suffering from adeficiency of the protein or who may benefit from increased levels ofthe protein. In some embodiments the cells are administered to thecirculatory system, e.g., by infusion. In some embodiments the cells areadministered intravenously. For example, hematopoietic cells, e.g.,RBCs, WBCs, platelets, may be administered to the circulatory system. Insome embodiments the cells are administered to the lymphatic system,e.g., by infusion. In some embodiments the protein is one that isnormally present in the blood, e.g., a protein produced by the liver orby hematopoietic cells or endothelial cells. In some embodiments theprotein is an enzyme. The enzyme may be catalytically active or maybecome catalytically active after administration. In some embodimentsthe enzyme may act on a substrate in the blood. In some embodiments atleast a portion of the protein attached to sortagged may be releasedfrom the cells in vivo. Release may occur via cleavage that, in someembodiments, also activates the protein. For example, a protein may beattached as an inactive enzyme precursor (zymogen). In some embodimentssortase-modified cells are administered locally, e.g., to a tissue ororgan in which a protein or a substrate of the protein is normallyproduced or active. Examples of various diseases associated withdeficiency of various proteins, e.g., enzymes, are provided above.

In certain embodiments mammalian cells conjugated with an anti-microbialagent and/or with a targeting moiety that targets the cells to a microbeor parasite may be administered to a subject who is at risk of infectionor is infected by the microbe or parasite, e.g., a subject who has beenrecently exposed to the microbe or parasite, has been determined toharbor detectable levels of the microbe or parasite (e.g., in theblood), or is clinically ill with an infection caused by the microbe orparasite. The cells may be administered to the circulatory system orlocally at or near a site of infection.

In certain embodiments mammalian cells conjugated with an agent thatinhibits a toxic or harmful substance maybe administered to a subjectwho has been exposed to the toxic or harmful substance and is at risk oftoxicity or damage from the substance, a subject who exhibits symptomsof exposure, or a subject who is at risk of exposure. For example, asubject who is infected by or has been exposed to a toxin-producingpathogen that may be treated with cells that have been conjugated withan agent that inhibits the toxin.

In some aspects, sortase is used to modify mammalian cells that may beused in a cell based immunotherapy. For example, in some embodiments oneor more epitopes or antigens (e.g., epitopes or antigens of pathogens ortumors) may be conjugated to mammalian cells using sortase. In someembodiments the mammalian cells are hematopoietic cells, e.g., RBCs. Insome embodiments the mammalian cells are fibroblasts. In someembodiments, the sortase-modified cells may be administered to a subjectin order to induce or enhance an immune response against the antigen orcells comprising the antigen(s), e.g., pathogens, pathogen-infectedcells, tumor cells, toxic substances, etc. In some embodiments, thesortase-modified cells may be used ex vivo, e.g., to stimulate oractivate other cells, e.g., immune system cells such as T cells,antigen-presenting cells (e.g., dendritic cells, macrophages), B cells,NK cells, or precursors of any such cells. In some aspects, the presentdisclosure provides methods of producing or modifying a cellularartificial APC. For example, in some embodiments, a cell is sortaggedwith one or more moieties (e.g., antigens, TCR-engaging molecules,costimulatory molecules) that allows it to serve as an aAPC or improvesits ability to serve as an aAPC, e.g., to provide costimulation. In someembodiments cells that have been stimulated ex vivo by contact with asortase-modified cell are administered to a subject, e.g., to induce orenhance an immune response directed to a pathogen, pathogen-infectedcells, tumor, toxic substance, etc.

In some embodiments, sortase-modified mammalian immune system cells areused in adoptive immunotherapy. “Adoptive immunotherapy”, also called“adoptive cell transfer” (ACT) refers to administration (transfer) ofimmune system cells to a subject, e.g., for therapeutic purposes. Insome embodiments administered immune system cells exert an immuneresponse against a tumor, pathogen, or pathogen-infected cells. Forexample, cytotoxic T cells (e.g., CD8+ T cells), NK cells, or phagocytes(e.g., neutrophils, macrophages) may exert direct cytotoxic effects ontarget cells. In some embodiments target cells may be tumor cells,tumor-associated cells, pathogens, pathogen-infected cells, or otherunwanted cells. In some embodiments target cells may be immune systemcells that exert an abnormal or damaging effect on self cells ortissues. For example, target cells may be autoreactive T cell clones,plasma cells that produce autoantibodies, etc.

In certain embodiments immune system cells (e.g., cytotoxic T cells, NKcells, CD4+ T cells, DCs, neutrophils, macrophages) are sortagged with atargeting moiety that targets the cells to a tumor cell,tumor-associated cell, pathogen, pathogen-infected cell, or otherunwanted target. The targeting moiety may be any moiety that binds tothe target. The targeting moiety may, for example, comprise an antibody,antibody fragment, engineered polypeptide, aptamer, or ligand (e.g.,small molecule ligand). In some embodiments the targeting moiety bindsto a tumor antigen. The targeting moiety targets the cell to tumor cellsthat express the tumor antigen. Binding agents (e.g., monoclonalantibodies) that specifically bind to a variety of different tumorantigens are known in the art, and additional binding agents can beidentified using methods known in the art. For example, trastuzumabbinds to Her2/Neu and may be used as a targeting moiety to target tumorsthat express Her2/Neu (e.g., certain breast cancers). Rituximab binds toCD20 and may be used as a targeting moiety to target malignancies thatexpress CD20 (e.g., non-Hodgkin's lymphoma (e.g., follicular lymphoma),diffuse large B cell lymphoma, mantle cell lymphoma). In certainembodiments the sortagged cells mount an immune response or promote anendogenous immune response against tumor cells, tumor-associated cells,pathogens, pathogen-infected cells, or other unwanted cells orsubstances to which they are targeted. The immune response may comprisecell-mediated cytotoxicity, antibody production, release of cytokines orother agents that damage the target cells, etc. In some embodiments theadministered cells may have been exposed in vitro to one or moreantigens expressed by a tumor cell, tumor-associated cell, pathogen, orpathogen-infected cell. The cells may be exposed prior to or aftersortagging. In some embodiments the cells may have been exposed ex vivoto APCs that present an epitope of an antigen expressed by a tumor cell,tumor-associated cell, pathogen, or pathogen-infected cell. In someembodiments the cells are exposed in vitro to agents that stimulate thecells to proliferate, differentiate, or become activated, such asanti-CD3 antibodies, co-stimulators, etc. In some embodiments, targetingAPCs, e.g., DCs, to a tumor, pathogen, pathogen-infected cell, or othertarget results in increased presentation of target-associated antigensby the APCs, which may enhance an immune response mounted by endogenousor administered immune system cells. In some embodiments, targeting CD4+T cells to a tumor, pathogen, pathogen-infected cell, or other targetresults in increased provision of “helper” functions by such cells,which may enhance an immune response mounted by endogenous oradministered immune system cells that receive such help.

In some embodiments mammalian cells are sortagged with an agentcomprising a substance that is capable of exerting toxic effects on atarget cell, e.g., a tumor cell, tumor-associated cell, pathogen, orpathogen-infected cell. In some embodiments the cells are administeredto a subject and deliver the toxic substance to a target such as atumor. In some embodiments the toxic substance comprises a pro-apoptoticagent, a cytolytic agent, a cytotoxic drug, or a toxin. In someembodiments the target cells are relatively susceptible to the toxiceffect of the toxic substance as compared with the sortagged cells. Forexample, a sortagged cell may on average remain viable when sortaggedwith the toxic substance and able to deliver the toxic substance to atarget cell in an amount that is lethal to the target cell. The targetcells may differ from the sortagged cells in one or more ways thatrenders the target cells more susceptible to effects of the toxicsubstance. Differences in tumor cell metabolism, proliferation rate, oroncogene expression, as compared with most non-tumor cells may beexploited to select agents that are selectively toxic to tumor cells. Insome embodiments the sortagged cells may either naturally or as a resultof engineering lack a receptor for the toxic substance or may lack amolecular target or functional biological pathway by which the toxicsubstance acts or otherwise be rendered relatively less susceptible toeffects of the toxic substance.

In some embodiments mammalian cells, e.g., immune system cells, may besortagged with an agent comprising a cytolytic domain. For example,cells may be sortagged with a perforin, granzyme, granulysin orbiologically active domain thereof. Such a cytolytic domain may increasethe cytotoxic activity of the cells (e.g., if the cells are cytotoxiccells) or may confer cytotoxic activity on cells that would otherwiselack it. In some embodiments a cytolytic domain comprises or is derivedfrom a cytolytic agent that is naturally found in a subject, e.g., onethat is normally used by immune system cells of the subject to lysecells. In some embodiments a cytolytic domain comprises or is derivedfrom a cytolytic agent that is not naturally found in a subject. Forexample, a variety of cytolytic toxins, e.g., cytolysins produced byvarious microbes, are known (see, e.g., discussion below). An agent mayfurther comprise a targeting moiety that targets the cells to targetcells to be lysed, e.g., tumor cells, infected cells, pathogens. In someembodiments a targeting moiety is separately conjugated to the cells.

In some embodiments mammalian cells, e.g., immune system cells, may besortagged with a pro-apoptotic agent. In some embodiments apro-apoptotic agent is an agent comprising a domain that promotesapoptosis (pro-apoptotic domain). In some embodiments a pro-apoptoticagent is an agent that can initiate or enhance an apoptotic pathway ifintroduced into or activated in a cell. As known in the art, apoptosisis a process of programmed cell death that occurs in multicellularorganisms, e.g., mammals. A pro-apoptotic domain may be any domain thatcan deliver an extracellular signal that initiates or enhances anapoptotic pathway. In some embodiments an apoptotic pathway leads toactivation of caspases, e.g., initiator caspases, which in turn activateeffector caspases. Effector caspases proteolytically degrade a varietyof intracellular proteins to carry out the cell death program.Pro-apoptotic domains include, e.g., ligands that bind to TNF alphareceptors (e.g., TNF-R1) or Fas in mammalian cells. In some embodimentsa pro-apoptotic domain may comprise any moiety that can activate TNF-R1or Fas. In some embodiments a pro-apoptotic domain comprises FasL,Trail, Tweak, Lymphotoxin, TNF alpha, or a biologically active domainthereof. A pro-apoptotic domain or other pro-apoptotic agent mayincrease the cytotoxic activity of the cells (e.g., if the cells arecytotoxic cells) or may confer cytotoxic activity on cells that wouldotherwise lack it. In some embodiments a pro-apoptotic domain or otherpro-apoptotic agent comprises or is derived from a protein that isnaturally found in a subject, e.g., one that is normally used by immunesystem cells of the subject or during development or other physiologicalprocesses that involve apoptosis. In some embodiments a pro-apoptoticdomain or other pro-apoptotic agent comprises or is derived from asubstance that is not naturally found in a subject. In some embodimentsa pro-apoptotic agent comprises a TRAIL receptor agonist. TRAILreceptors include TRAIL-R1 (also known as death receptor 4 (DR4) andTRAIL-R2 (also known as death receptor 5 (DR5)). TRAIL receptor agonistsinclude the natural ligand TRAIL and other agents that bind to a TRAILreceptor and mimic the effect of TRAIL. In some embodiments the TRAILreceptor agonist is a monoclonal antibody that binds to TRAIL-R1 and/orTRAIL-R2, such as Mapatumumab (human anti-DR4 mAb), Tigatuzumab(humanized anti-DR5 mAb), Lexatumumab (human anti-DR5 mAb), Conatumumab(human anti-DR5 mAb), Apomab (human anti-DR5 mAb). In some embodimentsTRAIL or a TRAIL receptor agonist is useful in treatment of breast,colon, lung, pancreatic, prostate, renal carcinoma, thyroid carcinoma,glioma, multiple myeloma or leukemia. In some embodiments apro-apoptotic agent induces apoptosis by inhibiting an anti-apoptoticprotein such as Bc1-2, Bc1-xL, and Bcl-w or by activating apro-apoptotic protein such as BAX, BID, BAK, or BAD inside a cell. Forexample, a pro-apoptotic agent may be a member of the BH3-only family ofproteins or a biologically active domain thereof. BH3-only proteinsinhibit antiapoptotic members of the Bcl-2 family. Examples of suchproteins include NOXA or a biologically active fragment thereof (e.g.,the BH3 domain or the mitochondrial targeting domain). In someembodiments the pro-apoptotic agent comprises a small molecule BH3mimetic such as ABT-737, ABT-263 (navitoclax) or ABT-199 (Vandenberg C Jand Cory, S., Blood. 2013; 121(12):2285-8 and references therein). Insome embodiments, a pro-apoptotic domain is useful to treat a cancerthat has increased expression or activity of an anti-apoptotic proteinas compared with non-cancer cells. In some embodiments, a pro-apoptoticdomain is useful to treat a cancer that has become resistant tocytotoxic chemotherapy or to inhibit emergence of such resistance. Apro-apoptotic agent may further comprise a targeting moiety that targetsthe cells to target cells whose apoptosis is desired e.g., tumor cells,infected cells. In some embodiments a targeting moiety is separatelyconjugated to the cells.

In certain embodiments mammalian cells, e.g., immune system cells, aresortagged with an immunomodulator. In some embodiments theimmunomodulator promotes survival, proliferation, differentiation and/orone or more activities of at least some of the administered cells. Insome embodiments the immunomodulator comprises a stimulatory cytokine,costimulatory molecule (e.g., OX40, OX40L, CD137L), or adjuvant (e.g., aCD40 ligand, anti-CD40 antibody, TLR ligand). In certain embodimentsimmune system cells are sortagged with a targeting moiety and animmunomodulator. A targeting moiety and an immunomodulator may beprovided as individual agents or may be part of a bifunctional agent.

In some embodiments administered immune system cells may modulate immunesystem cells that are co-administered to the subject or already presentin the subject. For example, administered immune system cells maystimulate or inhibit proliferation, activation, differentiation,migration, and/or maturation of immune system cells present in thesubject. Stimulation or inhibition may result at least in part fromsecretion of cytokine(s) by the transferred cells and/or from cell-cellinteractions (e.g., display of costimulatory molecules or inhibitorymolecules such as CD28/CTLA-4 family members (e.g., CTLA-4 or PD-1ligand) by the transferred cells). In some embodiments a moietyconjugated to immune system cells may comprise a costimulator. Suchcells may provide costimulation at a tumor site or site of infection,which may, for example, enhance the ability of tumor-specific orpathogen-specific T cells to eliminate tumor cells or infected cells.Whether transferred cells stimulate or inhibit the immune system of thesubject may depend on a variety of factors, such as the properties ofthe particular cell type transferred and/or the identity and/or amountof the agent conjugated to the transferred cells. Such factors may beselected according to the desired effect of the cells. Transferred CD4+T cells may provide “help” to endogenous or co-administered cytotoxiccells, which may expand and/or augment the ability of cytotoxic cells toeliminate target cells. Transferred Treg cells may suppress immuneresponses of endogenous immune system cells that may otherwise exertunwanted or deleterious activity against the subject's own cells ortissues or against transplanted cells or tissues. Such suppression maybe useful to treat autoimmune diseases or reduce the likelihood ofrejection of a transplant.

In some embodiments immune system cells comprise a polyclonalpopulation, in that the population comprises multiple subpopulations ofcells that express TCRs or BCRs that are specific for a variety ofdifferent targets, antigens, or epitopes. For example, a polyclonalpopulation may comprise T and/or B cells that collectively express atleast 10³, 10⁴, 10⁵, or more distinct TCRs or BCRs. A polyclonalpopulation of lymphocytes may be obtained, e.g., from peripheral blood.In some embodiments immune system cells comprise a monoclonal populationof T or B cells in that cells in the population carry TCRs or BCRs thatare specific for a particular epitope, which may be an epitope presentin or on a target such as a tumor cell, tumor-associated cell, pathogen,pathogen-infected cell, or other unwanted cell. In some embodiments twoor more monoclonal populations may be combined, e.g., populations havingTCRs or BCRs specific for different epitopes of a target antigen ordifferent antigens of a target entity.

In some embodiments autologous or allogeneic T cells with anti-tumoractivity are obtained, optionally expanded and/or activated in vitro,and sortagged, (e.g., with a targeting moiety, biologically activemoiety, or both). In some embodiments the sortagged T cells areintroduced into a subject in need of treatment for cancer. In someembodiments T cells comprise autologous tumor-infiltrating lymphocytes(TILs). Autologous TILs may be obtained using methods known in the artfrom a tumor following biopsy or removal of the tumor from the subject.In some embodiments allogeneic T cells are a T cell line, e.g., theNK-92 cell line or a derivative thereof.

In some embodiments autologous or allogeneic NK cells with anti-tumoractivity are obtained, optionally expanded and/or activated in vitro,and sortagged, (e.g., with a targeting moiety, biologically activemoiety, or both). In some embodiments the sortagged NK cells areintroduced into a subject in need of treatment for cancer. In someembodiments allogeneic NK cells are an NK cell line, e.g., the NK-92cell line or a derivative thereof.

In some embodiments immune system cells may be contacted in vitro withone or more epitopes, e.g., in order to activate cells that recognizesuch epitope(s). In some embodiments immune system cells may becontacted in vitro with microbes or parasites, tumor cells, tumortissue, pathogen-infected cells, cells of a tumor cell line, material(e.g., proteins, RNA, membrane fraction, lysate) derived from such cellsor tissue, or partly purified or synthetic antigens or epitopes (e.g., apeptide pool). In some embodiments immune system cells (e.g., T cells)that bind to or proliferate in response to such cells or substances maybe isolated. The immune system cells (e.g., T cells) may be sortagged,e.g., with a targeting moiety that targets them to a tumor or pathogen,and administered to a subject who is in need of treatment of a tumor orwho is infected or at risk of infection by the pathogen. In someembodiments the cells or substances used to stimulate or isolate theimmune system cells are derived from a particular patient's tumor orcomprise TA(s) or TA epitopes found in a particular patient's tumor (ortypically found in tumors of that type), and the sortagged immune systemcells are administered to the patient.

In some embodiments a method may comprise isolating or determining theidentity of one or more antigens or epitopes expressed by tumor cells ortumor-associated cells obtained or originating from a subject andconjugating a targeting moiety that binds to at least one of theantigens or epitopes to mammalian cells in vitro. The antigens orepitopes may be identified or isolated using any of a variety of methodsused in the art. RNA (e.g., mRNA) or proteins from tumors, tumor cells,or tumor tissue samples can be analyzed using standard methods for RNAor protein detection and quantification. For example, proteins may beanalyzed using immunological methods such as immunohistochemistry orELISA. In some embodiments the cells comprise immune system cells, e.g.,cytotoxic cells, e.g., cytotoxic T cells or NK cells. In someembodiments a cytotoxic cell releases proteins (e.g., cytolyticproteins) that induce lysis of a target cell. In some embodiments acytotoxic cell is able to induce apoptosis in a target cell. In someembodiments the cells comprise T helper cells. The method may furthercomprise administering the cells to the subject. In some embodiments amethod may comprise isolating or determining the identity of one or moreantigens expressed by tumor cells or tumor-associated cells obtained ororiginating from a subject, and conjugating a targeting moiety thatbinds to at least one of the antigen(s) to immune system cells ex vivo.The method may further comprise administering the cells to the subject.

In some embodiments, a tumor sample is analyzed to identify one or moreTAs expressed by the tumor, to which therapeutic cells are to betargeted. The tumor sample may be from a tumor removed at surgery, froma biopsy, blood sample (e.g., cells of a hematologic malignancy orcirculating tumor cells from a solid tumor). A panel of antibodies orother binding agents may be used to identify cell surface TAs. In someembodiments, a patient who has been treated for a tumor or is suspectedof having a tumor or tumor recurrence may be monitored by performingperiodic blood tests to detect a soluble tumor antigen or circulatingtumor cells. If test results show an increase or abnormally high levelof the soluble TA or presence of circulating tumor cells, a therapeuticcell composition of the present invention, comprising cells targeted toa TA, may be administered. For example, a patient who has been or isbeing treated for a tumor that expresses CA-125 may be monitored todetect increased blood levels of CA-125. If such increased levels aredetected, a therapeutic cell composition comprising cells targeted toCA-125 and/or targeted to a different TA expressed by the tumor may beadministered.

In some embodiments, cells are conjugated to a first targeting moietythat binds to a first tumor antigen and a second targeting moiety thatbinds to a second tumor antigen, wherein the first and second tumorantigens are expressed by cells of the same tumor (tumor cells and/ortumor associated cells). The first and second TAs may be expressed bydifferent cell populations in the tumor or may be expressed on at leastsome of the same cells of the tumor. The targeting moieties may be partof the same agent, e.g., a bispecific antibody, or may be two separateagents. In some embodiments, two cell populations are admininstered to asubject in need of treatment for a tumor: a first cell populationtargeted to a first TA and a second cell population targeted to a secondTA. For example, as noted above, certain tumors express CA125 andmesothelin. Cells to be administered to a subject in need of treatmentfor a tumor may be conjugated to a first targeting moiety that binds toCA125 and a second targeting moiety that binds to mesothelin or a firstcell population targeted to mesothelin and a second cell populationtargeted to CA125 may be administered. In some embodiments,administration of a cell targeted to two or more different tumorantigens may have an additive or greater than additive effect. In someembodiments, administration of two cell populations, each targeted to adifferent tumor antigen, may have an additive or greater than additiveeffect.

In some embodiments, cells, e.g., immune system cells, are sortaggedwith an agent comprising a targeting moiety that targets the cells tocirculating tumor cells (CTCs). CTCs are cells from a tumor (e.g., asolid tumor) that intravasate into the circulation (vascular systemand/or lymphatic system). CTCs may extravasate at sites to which theyare carried by the circulation, where they may survive and formmetastases. CTCs can interact with receptors on endothelial cell wallsin a way that resembles leukocyte extravasation in inflammation andlymphocyte homing. CTCs from many types of primary tumors expresssialylated carbohydrate ligands similar to those of leukocytes, whichmediate interactions with selectins on the endothelium. In someembodiments a selectin is used as a targeting moiety to target cells,e.g., immune system cells, to circulating tumor cells (CTCs). In someembodiments the cells are sortagged both with a selectin and with one ormore additional agents. In some embodiments the one or more additionalagents comprise a cytotoxic moiety, a detectable moiety, a secondtargeting moiety (e.g., a targeting moiety that binds to a TA expressedby the tumor), or any combination thereof. In some embodiments thecytotoxic moiety comprises a pro-apoptotic agent, e.g., a pro-apoptoticprotein such as TRAIL or a biologically active portion thereof or aTRAIL receptor agonist.

In some embodiments cells sortagged with a detectable moiety andtargeted to CTCs may be useful to detect the presence of CTCs in asample (e.g., a blood sample) obtained from a subject or in vivo. Thesubject may be suspected of having cancer or may have been treated forcancer. Without wishing to be bound by any theory, the fact that a cellmay be sortagged with numerous individual molecules of a detectablemoiety may facilitate detection of CTCs, e.g., by making detection morereliable (e.g., fewer false positive and/or false negative results)and/or by permitting detection of smaller numbers of CTCs than withvarious other methods.

Cancer stem cells (CSCs) are cancer cells found within tumors orhematological cancers that possess characteristics analogous tocharacteristics associated with normal stem cells. CSCs have thecapacity to initiate tumors, self-renew, and differentiate intophenotypically diverse cancer cells. CSCs are often relatively resistantto chemotherapy drugs and radiation and are proposed to persist andcause relapse and metastasis by giving rise to new tumors. CSCs mayexpress a variety of cell surface markers at levels that differentiatethem from non-CSC cancer cells and/or most normal cells. For example,CD133, CD44, and EpCAM have been identified as CSC markers in a varietyof epithelial cancers. In some embodiments a binding moiety, e.g., anantibody, that binds to a CSC marker is used as a targeting moiety totarget cells, e.g., immune system cells, to CSCs. In some embodimentsthe cells are sortagged both with a CSC marker and with one or moreadditional agents. In some embodiments the one or more additional agentscomprise a cytotoxic moiety, a detectable moiety, a second targetingmoiety (e.g., a targeting moiety that binds to a TA expressed by non-CSCtumor cells or tumor-associated cells), or any combination thereof. Insome embodiments the cytotoxic moiety comprises a pro-apoptotic agent,e.g., a pro-apoptotic protein such as TRAIL or a biologically activeportion thereof or a TRAIL receptor agonist.

In some embodiments APCs are contacted in vitro with tumor cells, tumortissue, pathogen-infected cells, material (e.g., proteins, RNA, membranefraction, lysate) derived from such cells or tissue, or partly purifiedor synthetic antigens or epitopes (e.g., a peptide pool) and are thenused to stimulate lymphocytes in vitro. In some embodiments thestimulated lymphocytes may be sortagged (e.g., with a targeting moietythat targets them to a tumor, with a chemotherapeutic agent potentiallyactive against the tumor, and/or with an immunomodulator) and may beadministered to a subject who is in need of treatment of a tumor or isat risk of developing a tumor or of tumor recurrence. In someembodiments the stimulated lymphocytes may be sortagged (e.g., with atargeting moiety that targets them to a pathogen or pathogen-infectedcell, with an antimicrobial agent potentially active against thepathogen, and/or with an immunomodulator) and may be administered to asubject who is in need of treatment of an infection or is at risk ofinfection by the pathogen.

Protocols for T cell activation and/or expansion may include, e.g.,culturing the cells in medium containing appropriate cytokines such asinterleukin-2 (IL-2) and/or appropriate co-stimulatory molecules. Insome embodiments an expansion protocol using IL-2 and CD3 ligation viaan anti-CD3 antibody may be used (so-called “rapid expansion method”,e.g., as described in Dudley M E, et al., Generation oftumor-infiltrating lymphocyte cultures for use in adoptive transfertherapy for melanoma patients. J Immunother 2003, 26:332-342). Thecytokine(s) may be provided as isolated proteins or by co-culture withcells that secrete them either naturally or as a result of geneticmanipulation. In some embodiments cells are cultured in the presence ofanti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, theantibodies are attached to beads (e.g., paramagnetic beads) or anothersupport, such as the interior sides and bottom of a cell culture vessel.In some embodiments, a mixture of anti-CD3 and anti-CD28 antibodies isattached to the beads (coimmobilization) (see, e.g., Levine, B., et al.,Tumaini, B., et al. (both cited above), and various other referencescited herein. In some embodiments, cells are co-cultured with PBMCs,which comprise cells that provide one or more molecules that promoteactivation and/or expansion. In some embodiments cells are cultured withPBMC and anti-CD28 antibodies (e.g., attached to a support such asbeads). In some embodiments the PBMCs are immunocompatible with the Tcells. In some embodiments both the T cells and PBMCs are derived from asubject to whom the T cells are to be introduced. In some embodiments aratio of beads to cells between 100:1 and 1:100 is used. In someembodiments, a ratio of beads to cells between 10:1 and 1:10, between5:1 and 1; 5, between 3:1 and 1:3, or about 1:1 is used.

In some embodiments lymphocytes are activated in vitro by contactingthem with APCs. In some embodiments artificial APCs may be used. Avariety of cellular aAPCs are known in the art. For example, K562 cells(available from the ATCC, Manassas, Va.) can serve as artificial APC.Such cells have been engineered to express a variety of co-stimulatorymolecules and used for ex vivo expansion of polyclonal andantigen-specific cytotoxic T lymphocytes, natural killer cells, andantigen-experienced tumor-infiltrating lymphocytes (Maus M V, et al. NatBiotechnol 2002, 20:143-148; Suhoski M M, et al. Mol Ther 2007,15:981-988; Fujisaki, H., Cancer Res 2009; 69: 4010-4017; Ye Q, et al.,J Transl Med 2011, 9:131; Butler, M O, et al., Clin Cancer Res Mar. 15,2007 13; 1857). Cellular aAPC (e.g., a cellular aAPC that has beengenetically engineered and/or has been or is to be sortagged) may beirradiated (e.g., with gamma radiation, e.g., about 100 Gy in certainembodiments) or otherwise rendered unable to proliferate. In someembodiments noncellular aAPCs may be used. Noncellular aAPCs may beparticles that have antigen presenting molecules (APMs) attachedthereto. Such particles may be contacted with antigen or antigenfragment (e.g., peptides) to allow them to serve as aAPCs. In someembodiments, noncellular APCs may be particles such as liposomes, beads(e.g., paramagnetic beads), particles comprised at least in part oforganic polymers, quantum dots, etc. Such particles may have any of avariety of different molecules attached to their cell surface, e.g., anyof the molecules discussed above in reference to cellular aAPC.Noncellular aAPCs may be generated as described in Oelke M, et al., Exvivo induction and expansion of antigen-specific cytotoxic T cells byHLA-Ig-coated artificial antigen-presenting cells. Nat Med. 2003,9:619-625; Webb, T, et. al., J Immunol Methods, 2009, 346(1-2): 38-44;Oelke M. and Schneck, J P, Immunol Res (2010); 47:248-256; East, J E, JVis Exp. Artificial antigen presenting cell (aAPC) mediated activationand expansion of natural killer T cells. 2012 Dec. 29; (70). pii: 4333.doi: 10.3791/4333. In some embodiments APMs may be conjugated to asupport, e.g., beads (e.g., magnetic beads), inner surface of a vesselsuch as a well or plate, etc. APCs may be loaded with antigen prior toor after being conjugated to a support. Cells to which the antigen is tobe presented are contacted with the support to permit antigenpresentation to occur. In some embodiments an APM may be conjugated orfused to an IgG domain or other moiety that may serve as a linker toattach the APM to a support.

In some embodiments lymphocytes, e.g., T cells, are stimulated in vitroby co-culturing them with APCs, e.g., DCs or aAPCs. In some embodimentsthe APCs display at least one epitope that stimulates proliferationand/or effector activity of T cells that bind to it. In some embodimentsan agent comprising one or more epitopes is conjugated to the APCs,e.g., using sortase. In some embodiments an agent comprising one or moreepitopes is non-covalently bound to a cell surface molecule or complexexpressed by APCs. In some embodiments the cell-surface molecule orcomplex is Dec-205, a C type lectin such as DC-SIGN, or MHC class II(MHCII) protein. In some embodiments the cells have been sortagged orthe cells and/or descendants thereof are subsequently sortaggedaccording to methods described herein. In some embodiments sortaggedmammalian cells are administered to a subject.

In some embodiments a moiety conjugated to immune system cells usingsortase may inhibit or overcome mechanisms that may exist in a subjectthat may otherwise limit the therapeutic efficacy of cellularimmunotherapy or may limit the effectiveness of the subject's endogenousimmune system. In the case of therapy for cancer, such mechanisms mayinclude immune evasion mediated by the secretion of immunosuppressivesubstances such as transforming growth factor β (TGFβ) in themicroenvironment of a tumor and/or mediated by the accumulation ofregulatory T cells, both of which can, for example, dampen the in vivoactivation, expansion, and tumor homing of transferred tumor-reactiveCD8+ T cells. In the case of therapy for diseases caused by pathogens,such mechanisms may include any of various immunoevasive orimmunosuppressive agents produced or encoded or induced by pathogens. Amoiety conjugated to cells may inhibit the effect of an immunoevasive orimmunsuppressive substance by, for example, binding to the substance,acting as an antagonist or competitor at a receptor for the substance,or antagonizing a pathway activated by the substance. In someembodiments a moiety conjugated to cells may inhibit development oractivity of Tregs that would otherwise dampen an immune response againsta tumor, pathogen, or pathogen-infected cell.

“Immune checkpoint pathways” or “immune checkpoints” are naturallyexisting inhibitory pathways of the immune system that play importantroles in maintaining self-tolerance and modulating the duration andlevel of effector output (e.g., in the case of T cells, the levels ofcytokine production, proliferation or target killing potential) ofphysiological immune responses in order to minimize damage to thetissues of the individual mounting the immune response. Such pathwaysmay, for example, downmodulate T cell activity or enhance regulatory Tcell immunosuppressive activity. Examples of immune checkpoint pathwaysinclude, e.g., the PD-1 pathway and the CTLA-4 pathway, discussedfurther below. Tumors frequently co-opt certain immune-checkpointpathways as a major mechanism of immune resistance, e.g., against Tcells that are specific for tumor antigens. Certain aspects of theinvention utilize sortase-modified cells to inhibit immune checkpointmechanisms. In some aspects, the invention provides mammalian cellsconjugated using sortase to a moiety comprising an immune checkpointmodulator. In some embodiments the immune checkpoint modulator is animmune checkpoint inhibitor. “Immune checkpoint inhibitor” refers to anyagent that inhibits (suppresses, reduces activity of) an immunecheckpoint pathway. In some embodiments the immune checkpoint modulatoris an immune checkpoint activator. “Immune checkpoint activator” refersto any agent that activates (stimulates, increases activity of) animmune checkpoint pathway. In some embodiments the cells arenon-genetically modified cells. In some embodiments the cells arederived from a subject in need of treatment for cancer or animmunocompatible donor. In some embodiments the cells comprise PBMCs orRBCs. In some embodiments the cells comprise lymphocytes. “Immunecheckpoint protein” refers to those proteins that are components ofimmune checkpoint pathways, and include membrane-bound, soluble (e.g.,secreted), and intracellular proteins. Many immune checkpoint pathwaysare initiated by interactions between membrane-bound receptors andsoluble or membrane-bound ligands. In some embodiments an immunecheckpoint inhibitor is an agent that binds to receptor or ligand thatis a component of an immune checkpoint pathway. Binding of the agent tothe receptor or ligand blocks the ligand-receptor interaction, thusinhibiting the immune checkpoint pathway. For example, in someembodiments a moiety inhibits an interaction between PD-1 and a PD-1ligand, e.g., by binding to either PD-1 or PD-1 ligand.

In some embodiments an agent comprises a modulator of the PD-1 pathway.PD-1 is an inhibitory surface receptor expressed by a variety of cells,including activated T cells, B cells, natural killer T cells, monocytes,and dendritic cells (DC). PD-1 has two naturally occurring ligands,programmed cell death ligand 1 (PD-L1) and programmed cell death ligand2 (PD-L2), also called B7-H1 and B7-DC, respectively. The term “PD-L”refers to either or both PD-L1 and PD-L2. Where the term “PD-L” is usedherein, certain embodiments pertain to PD-L1, certain embodimentspertain to PD-L2, and certain embodiments pertain to both PD-L1 andPD-L2. The term “PD-1 pathway” refers to the biological processes thatoccur in a cell, e.g., an immune system cell, upon binding of PD-L toPD-1 expressed by the cell. Binding of PD-L to PD-1 on immune systemcells, e.g., helper or cytotoxic T cells, typically has inhibitoryeffects on their proliferation and/or activity, particularly in thecontext of stimulation by antigen, and may contribute to T cellexhaustion. However, the PD-1 pathway can promote the development andactivity of T regulatory cells, which may suppress the activity of otherimmune system cells (e.g., helper and/or cytotoxic T cells) that wouldotherwise mount an immune response, e.g., against a tumor, infectedcell, pathogen, or a self antigen (e.g., in a subject with an autoimmunedisease). Tumors can use the PD-1 pathway to inhibit immune system cellsthat may otherwise mount an immune response against the tumor. Certaintumors express PD-L1, and its expression has been correlated with tumoraggressiveness and inversely correlated with survival of patients,possibly because natural antitumor immunity against such tumors isinhibited. The PD-1 pathway has also been shown to impair immunefunction in various infections such as influenza virus infection, HIVinfection, HCV infection, and Mycobacterial infection. In someembodiments, inhibiting the PD-1 pathway is of use in treatment ofconditions in which the PD-1 pathway inhibits the immune response ofadministered immune system cells (or their descendants) or endogenousimmune system cells against a tumor, pathogen, or infected cell and/orin which activity of the PD-1 pathway is abnormally or inappropriatelyhigh or in which there is an abnormally or inappropriately high numberand/or activity of Tregs. In some embodiments, activating the PD-1pathway is of use in treatment of conditions in which activity of thePD-1 pathway is abnormally or inappropriately low, conditions in whichthere is an abnormally low number and/or activity of Tregs, and/orconditions in which increased number or activity of Tregs may bebeneficial, e.g., in subjects at risk of or suffering from an autoimmunediseases, GVHD, and/or transplant rejection. A number of cells or levelof activity may be abnormally or inappropriately high or low in absoluteterms or relative to the number or activity of immune systems cells ofone or more other types or subtype, e.g., Th1, Th2, Th17, cytotoxic Tcells, helper T cells. In some embodiments, a modulator of the PD-1pathway is of use to restore an appropriate balance between Tregs andother immune system cells.

In some embodiments a modulator of the PD-1 pathway is an inhibitor ofthe PD-1 pathway. An inhibitor of the PD-1 pathway reduces activity ofthe PD-1 pathway as compared to the activity of the pathway in theabsence of the inhibitor. It will be understood that the effect of aninhibitor may be observable only in the presence of a ligand that would(in the absence of the inhibitor) bind to PD-1 and activate it. Aninhibitor of the PD-1 pathway may be referred to as a PD-1 antagonist.In some embodiments a PD-1 antagonist binds to PD-1 or PD-L and blocksthe interaction between PD-1 and PD-L. In some embodiments a PD-1antagonist comprises a nucleic acid (e.g., a nucleic acid aptamer),protein, peptide, or small molecule that binds to PD-1, PD-L1, or PD-L2,wherein if the PD-1 antagonist binds to PD-1, it does not significantlystimulate PD-1 signaling. In some embodiments, an anti-PD-1, anti-PD-L1,or anti-PD-L2 antibody may be used as a PD-1 antagonist. Nivolumab (afully human IgG4 monoclonal antibody), CT-011 (a humanized IgG1monoclonal antibody), and lambrolizumab (also known as MK-3475, ahumanized IgG4 monoclonal antibody) are examples of anti-PD-1antibodies. BMS-936559 (a fully human IgG4 monoclonal antibody),MPDL3280A (human monoclonal, Genentech), and MEDI4736 (Medimmune) areanti-PD-L1 antibodies.

In some embodiments, a PD-1 antagonist comprises at least a portion ofthe extracellular domain of PD-1 or a variant or fragment thereof thatbinds to PD-L. If a cell is genetically engineered to express a variantor fragment of PD-1, a variant or fragment that substantially lacks theability to inhibit cells that express it may be used. For example, thevariant or fragment may have a mutation or at least partial deletion ofits intracellular signaling domain. The variant or fragment may comprisethe transmembrane domain and, optionally, at least a portion of theintracellular domain or may comprise a transmembrane domain from adifferent protein or a synthetic transmembrane domain to cause it toremain attached to the cell surface rather than secreted as a solubleprotein. The extracellular domain of PD-1 or a variant or fragmentthereof may serve as a targeting moiety to target cells, e.g., T cellsor NK cells, to a tumor comprising cells that express PD-L (e.g.,PD-L1). By binding to PD-L at the surface of tumor cells ortumor-associated cells or infected cells that express PD-L, PD-1extracellular domain may alternately or additionally serve to block theeffect of PD-L on immune system cells that express PD-1. Thus, theimmune suppressive effects of PD-L, e.g., PD-L1, expressed in tumors orinfected cells, may be reduced. A variety of PD-1 antagonists, e.g.,antibodies that bind to PD-1, PD-L1, or PD-L2 are described in U.S. Pat.Pub. No. 20040213795, 20110195068, 20120039906, 20120114649,20130095098, 20130108651, 20130109843, 20130237580, and 20130291136,among others.

In some embodiments a modulator of the PD-1 pathway increases activityof the PD-1 pathway, e.g., by binding to and activating PD-1. Amodulator of the PD-1 pathway that binds to and activates PD-1 may bereferred to as a PD-1 agonist. A PD-1 agonist may comprise abiologically active fragment or variant of the extracellular domain ofPD-L1 or PD-L2 or may mimic the effect induced by binding of PD-L1 orPD-L2 to PD-1. Cells that are sortagged with a PD-1 agonist may beuseful in conditions in which it is desired to inhibit the immuneresponse.

An agent that binds to PD-1 or PD-L may serve as a targeting moiety. Forexample, an agent that binds to PD-L may target cells that express orare sortagged with the agent to cells that express PD-1. The agent mayalso serve as a PD-1 agonist or antagonist in various embodiments. Anagent that binds to PD-1 may target cells that express or are sortaggedwith the agent to cells that express PD-L. The agent may also serve as aPD-1 agonist or antagonist in various embodiments.

In some embodiments an agent comprises a modulator of a T cellco-inhibitory receptor, such as the anti-inflammatory receptor cytotoxicT lymphocyte antigen-4 (CTLA-4). CTLA-4 is an important negativeregulator of T cell activation. In some embodiments a modulator ofCTLA-4 inhibits CTLA-4 activity and may be referred to as a “CTLA-1antagonist”. Inhibiting negative regulation mediated by CTLA-4 has beenshown to promote stimulation of adaptive immunity and potentiation of Tcell activation. Binding of a CTLA-4 ligand (e.g., B7-1 or B7-2) toCTLA-4 can reduce T cell proliferation and functional activity andpromote tolerance, which may reduce the ability of administered immunesystem cells and/or endogenous immune system cells of a subject to mountan immune response against a tumor, pathogen, or infected cells.Accordingly, in some embodiments a CTLA-4 antagonist is useful to limitor prevent negative regulation by CTLA-4. In some embodiments, a CTLA-4antagonist comprises an agent that binds to CTLA-4 and blocks CTLA-4ligands from binding to CTLA-4. For example, anti-CTLA-4 antibodies,such as ipilimumab or tremelimumab, may be used. In some embodiments aCTLA-4 antagonist comprises an antibody that binds to a CTLA-4 ligand.In addition to CTLA-4, other T cell co-inhibitory receptors including B7family members B7-H3, B7-H4, T cell immunoglobulin and mucindomain-containing protein 3 (Tim-3), and lymphocyte activation gene-3(LAG-3), interact with their cognate ligands on various cells types,including APCs, regulatory T cells (Tregs), and nonhematopoietic cells,resulting in reduced T cell proliferation and functional activity. BTLAis an inhibitory receptor on T cells. Its ligand is herpesvirus entrymediator (HVEM), which is expressed on certain tumor cell types such asmelanoma and on tumor-associated endothelial cells.

In some embodiments, an agent inhibits production or immunosuppressiveeffect of adenosine. Such an agent may be referred to as ananti-adenosine agent. Adenosine is produced in the extracellularcompartment by two ectonucleotidases: CD39, which hydrolyzes ATP and ADPinto AMP, and CD73, which converts AMP into adenosine. Adenosine mayalso be released from dying cells, e.g., dying cells in a tumor. CD73 isexpressed on tumor cells and host immune system cells, including Tregsand myeloid-derived suppressor cells, and is known to inhibit T-cellproliferation and reduce cytokine production and cytotoxicity ofactivated T-cells via A2a receptor (A2aR) subtype activation, protectingthe tumour from immune-mediated destruction. Adenosine A2a receptor(A2aR), the ligand of which is adenosine (which may be released fromdying cells in a tumor), inhibits T cell responses in part by drivingCD4+ T cells to develop into Treg cells. In some embodiments ananti-adenosine agent is an inhibitor of CD73 or CD39. In someembodiments an anti-adenosine agent comprises an antibody or otherbinding moiety that binds to CD73 or CD39. Small molecules that areselective inhibitors of CD73 include, e.g., adenosine 5′-(α,β-methylene)diphosphate (APCP) and ZM241365. In some embodiments an anti-adenosineagent comprises an antibody or other binding moiety that binds to anA2aR and inhibits its activity by, e.g., blocking binding of adenosine.

In some embodiments, an agent that inhibits the biological activity ofany T cell co-inhibitory receptor may be conjugated to cells to reduceloss of T cell proliferation and/or preserve functional activity and/orinhibit any immune checkpoint pathway. For example, antibodies or otherbinding moieties that bind to B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3may be conjugated to cells to be administered to a subject, e.g., asubject with cancer. In some embodiments an antagonist of B7-H3, BTLA,A2aR, B7-H4, Tim-3, or LAG-3, such as antibodies or other bindingmoieties that bind to B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3 or to aligand of B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3 may be conjugated tocells to be administered to a subject, e.g., a subject with cancer. Forexample, the antibody or other binding moiety may bind to galectin-9(ligand of Tim-3), HVEM, or adenosine. In some embodiments cells aresortagged with two or more different immune checkpoint inhibitors. Theinhibitors may inhibit different immune checkpoint pathways. Forexample, cells may be sortagged with at least two inhibitors selectedfrom PD-1 antagonists, CTLA-4 antagonists, B7-H3 antagonists, BTLAantagonists, A2aR antagonists or other anti-adenosine agents, B7-H4antagonists, Tim-3 antagonists, or LAG-3 antagonists.

Cells may be sortagged with a mixture of agents, e.g., any of theafore-mentioned immune checkpoint inhibitor, or separate aliquots ofcells may each be sortagged with a single immune checkpoint inhibitorand administered in combination to a subject, e.g., a subject in need oftreatment for cancer. In some embodiments cells are sortagged with afirst immune checkpoint inhibitor and then with a second immunecheckpoint inhibitor or are sortagged with two or more immune checkpointinhibitors at the same time. In some embodiments cells are sortaggedwith a first immune checkpoint inhibitor and administered in combinationwith a second immune checkpoint inhibitor. For example, cells may besortagged with a PD-1 antagonist and administered in combination with aCTLA-4 antagonist not attached to cells. In some embodiments cells thathave been sortagged with an immune checkpoint inhibitor are administeredin combination with a targeted cancer therapy such as a protein kinaseinhibitor, e.g., a VEGF receptor inhibitor, RAF inhibitor, ALKinhibitor, EGF receptor inhibitor, ERB-B1 inhibitor, ERB-B2 inhibitor,FGF receptor inhibitor, PDGF receptor inhibitor, etc.

Without wishing to be bound by any theory, and without limiting theinvention in any way, the use of cells, e.g., RBCs or T cells, that areconjugated with a targeting moiety (e.g., an antibody) that binds to atumor antigen, or that express an antigen receptor (e.g., a chimericantigen receptor) that binds to a tumor antigen, to deliver one ormultiple immune checkpoint inhibitors, may more effectively direct theimmune checkpoint inhibitors to the tumor, increase local activity,and/or reduce unwanted systemic exposure and associated side effects ascompared, for example, with administering the immune checkpointinhibitors not attached to cells. For example, by combining an antibodydirected to a tumor associated antigen with one or more immunecheckpoint inhibitors, the checkpoint inhibitors may be more effectivelydirected to the tumor, increasing local activity, and reducing unwantedsystemic exposure and associated side effects. Again without wishing tobe bound by any theory, and without limiting the invention in any way,using cells, e.g., RBCs, T cells, NK cells, or others, that have beensortagged with agents such as bi-specific agents comprising one armdirected to a tumor associated antigen and another arm directed to CD3,to deliver such agents, the half-life of such agents may be extended,thereby improving their efficacy without increasing the amount orfrequency of dosing and/or may allow a decrease in the amount orfrequency of dosing without dimishing efficacy. In some embodiments thecells used to deliver a bispecific agent may be modified by sortase tocarry one or more antibodies to tumor associated antigens.

In some embodiments a subject who is to be treated with cells that havebeen sortagged with an immune checkpoint inhibitor has a cancer that hasbeen tested for expression of an immune checkpoint protein, e.g., areceptor or ligand that is a component of an immune checkpoint pathway.In some embodiments tumor cells, non-transformed cells in the tumorstroma, or both, express the immune checkpoint protein, or overexpressthe immune checkpoint protein, as compared to its expression in normalcells outside the tumor. In some embodiments, a tumor or tumor sample(e.g., a biopsy or surgical sample) is analyzed to identify one or moreimmune checkpoint proteins that are expressed or overexpressed relativeto normal cells by the tumor cells, non-transformed cells in the tumorstroma, or both. In some embodiments cells that are sortagged with aninhibitor of a particular immune checkpoint pathway that is identifiedas being active in a tumor are administered to the subject. In someembodiments cells that are sortagged with an antagonist of a particularimmune checkpoint protein that is identified as being expressed oroverexpressed in a tumor are administered to the subject. In someembodiments cells that are sortagged with an antagonist of a receptor orligand of a particular immune checkpoint protein that is identified asbeing expressed or overexpressed in a tumor may be administered to thesubject. In some embodiments a method comprises identifying an immunecheckpoint pathway that is active or an immune checkpoint protein thatis expressed in a tumor or tumor sample and administering cells that aresortagged with an agent that inhibits the immune checkpoint pathway orimmune checkpoint protein to a subject in need of treatment for thetumor. Expression of the immune checkpoint protein or activity of theimmune checkpoint pathway may be measured using any method known in theart. In some embodiments the level of mRNA encoding an immune checkpointprotein may be measured. In some embodiments the level of the proteinmay be measured, e.g., using an immunological assay such as an ELISAassay, immunohistochemistry, or other suitable methods.

In some embodiments a modulator of CTLA-4 activates CTLA-4 and may bereferred to as a “CTLA-4 agonist”. In some embodiments a CTLA-4 agonistis useful to reduce T cell proliferation and functional activity and/orpromote tolerance, e.g., in a subject at risk or suffering from anautoimmune disease, GVHD, or transplant rejection.

In some embodiments, an agent comprises an activator of CD137 (CD137agonist). CD137 (also known as 4-1BB), a member of the tumor necrosisfactor (TNF) receptor superfamily and is a T cell costimulator molecule.Certain anti-CD137 monoclonal antibodies activate CD137 and are able toactivate CD8+ T cells, causing them to produce interferon (IFN)-γ, andinduce cytolytic markers. BMS-663513 (Urelumab), a humanized anti-CD137antibody, is an example of a CD-137 agonist.

In some embodiments, cells are modified to comprise a targeting moietyand a second agent at their surface. Either the targeting moiety, secondagent, or both, may be conjugated to the cells using sortase. In someembodiments, the cells are genetically engineered to express thetargeting moiety and are conjugated with the second agent using sortase.In some embodiments, the cells are genetically engineered to express thesecond agent and are conjugated with the targeting moiety using sortase.In some embodiments, the targeting moiety, the second agent, or both,are conjugated via a sortase recognition sequence to an endogenous,non-genetically engineered polypeptide expressed by the cells. In someembodiments the cells are not genetically modified to express apolypeptide comprising an extracellular domain capable of serving as asortase substrate or nucleophile in a sortase-catalyzed reaction. Insome embodiments, the cells comprise a chimeric antigen receptor thatcomprises an antigen binding moiety that targets the cells to particulartarget cells, e.g., tumor cells, infected cells, or other undesiredcells. The second agent can, in general, comprise any of the variousagents described herein. In some embodiments the second agent comprisesa targeting moiety. In some embodiments the second agent comprises atleast a biologically active portion of a cytokine or chemokine. In someembodiments the second agent has a biological activity that enhances thebiological activity of the administered cells, e.g., against targetcells (e.g., tumor cells or infected cells) in the body of a subject. Insome embodiments the biological activity is anti-tumor activity,costimulatory activity towards endogenous immune system cells or towardsadministered immune system cells or their descendants, or inhibitoryactivity towards endogenous immune system cells that contribute to anundesired immune response such as in autoimmune disease or transplantrejection. In some embodiments the second agent inhibits a biologicalactivity that would otherwise inhibit or suppress activity of theadministered cells or of endogenous cells, e.g., against target cells(e.g., tumor cells or infected cells) in the body of a subject.

In some embodiments a targeting moiety binds to a molecule expressed intumor vasculature (a “tumor vasculature marker”). In some embodimentsthe tumor vasculature marker is overexpressed in tumor vasculature ascompared with normal vasculature. In some embodiments the tumorvasculature marker is expressed at the luminal surface of endothelialcells in tumor vasculature. In some embodiments the molecule expressedon tumor vasculature is PD-1, VEGFR1, VEGFR2, tumor endothelial marker 1(TEM1; also known as endosialin or CD248), or integrin αvβ3. In someembodiments the targeting moiety comprises one or more binding moieties,e.g., antibodies, that bind to PD-1, VEGFR1, VEGFR2, TEM1, or integrinαvβ3.

In some embodiments cells to be used in cancer therapy may be sortaggedwith an agent comprising a targeting moiety that binds to a tumorvasculature marker and administered to a subject. In some embodimentsthe cells are immune system cells, which in some embodiments may besortagged with a second agent, wherein the second agent has anti-tumoractivity, such as an immune checkpoint inhibitor. In some embodimentsthe cells are red blood cells that are sortagged with an agent that hasanti-tumor activity, such as an immune checkpoint inhibitor. In someembodiments the cells are CAR cells.

In some aspects the disclosure provides a bispecific agent comprising(a) a first binding moiety that binds to a molecule on a target cell;(b) a second binding moiety that binds to a molecule on an immune systemcell, e.g., a T cell, NK cell, or professional phagocyte (e.g., amonocyte, macrophage, or dendritic cell); and (c) a sortase recognitionmotif. In some embodiments the SRM is appropriately positioned to permitthe agent to participate in a sortase-catalyzed reaction. In someaspects the disclosure provides a mammalian cell, e.g., a human cell,sortagged with such a bispecific agent. In some embodiments the targetcell is a tumor cell, tumor-associated cell, pathogen-infected cell, orpathogen. In some embodiments the target cell is a tumor cell ortumor-associated cell, and the first binding moiety binds to a tumorantigen. In some embodiments the second moiety binds to a molecule onthe surface of a T cell, e.g., CD3 (e.g., CD3 delta chain, CD3 epsilonchain, CD3 gamma chain, CD3 zeta chain, or other components of theTCR-CD3 complex, e.g., TCR alpha or TCR beta chains) or CD28. In someembodiments the second binding moiety binds to a molecule on the surfaceof an NK cell, dendritic cell, or macrophage, e.g., an Fc receptor,e.g., an Fcγreceptor, e.g., FcγRI (CD64) or FcγRIIIA (CD16a). Othermolecules expressed at the surface of T cell, NK cells, and/orphagocytes are known to those of ordinary skill in the art.

In some embodiments the bispecific agent links the sortagged cell to theimmune system cell and to the target cell. The immune system cell isthereby linked to the target cell via the sortagged cell. In someembodiments, linking the immune system cell to the target cell via thesortagged cell causes one or more effector functions of the immunesystem cell (e.g., cytotoxicity) to be directed towards the target cell.Alternatively, or additionally, in certain embodiments in which thesortagged cell is an immune system cell, one or more effector functionsof the sortagged cell may be directed towards the target cell. In someembodiments, the individual or combined effect of attack by the immunesystem cell, the sortagged cell, or both, results in death of the targetcell. In some embodiments two or more immune system cells are linked tothe sortagged cell via the bispecific agents attached to the sortaggedcell. An individual sortagged cell may thus tether multiple immunesystem cells to a target cell. It should be noted that multiple targetcells may be located in close proximity to each other. For example,tumor cells are often located close to each other and/or close totumor-associated cells in a tumor. An immune system cell that is linkedto a target cell, e.g., via a sortagged cell, may attack other targetcells in close proximity to the target cell to which it is linked.

In some embodiments, binding of the second binding moiety to a moleculeon a T cell, NK cell, or phagocyte stimulates such cell. For example,binding of the second binding moiety to CD3 or CD28 on a T cell maystimulate the T cell to proliferate, become activated, secretecytokines, phagocytose, or exert cytotoxic effects on a target cell by,e.g., releasing cytotoxins and/or inducing apoptosis. Binding of thesecond binding moiety to an Fc receptor on an NK cell, dendritic cell,or macrophage may stimulate such cell. For example, an NK cell may bestimulated to exert cytotoxic effects on a target cell; a DC may bestimulated to secrete cytokines and/or provide cell-cell interactionsthat stimulate other immune system cells; a macrophage may be stimulatedto phagocytose a target cell. In some embodiments the sortagged cellstimulates the immune system cell to which it is bound, or vice versa.For example, either cell may secrete one or more cytokines or providestimulation via cell-cell contact (e.g., one cell may express a ligandfor a costimulatory receptor on the other cell). In some embodiments thesortagged cell is also sortagged with an agent that stimulates theimmune system cell or inhibits immunosuppression of the immune systemcell. In some embodiments, activation is at least in part dependent onthe presence of cells expressing the target antigen to which the firstbinding moiety binds. For example, stimulation may occur when the Tcell, NK cell, or phagocyte and the sortagged cell are localized at thesite of a target cell. In some embodiments the sortagged cell issortagged with two or more distinct bispecific agents, wherein thesecond binding moieties of the distinct bispecific agent differ inregard to the cell type or molecule to which they bind. An individualsortagged cell may thus bind to cells of two or more different celltypes, e.g., a T cell and a dendritic cell, or a T cell and amacrophage, in addition to a target cell.

In some aspects the disclosure provides a trivalent agent comprising (a)a first binding moiety that binds to a molecule on a target cell; (b) asecond binding moiety that binds to a molecule on an immune system cell,e.g., a T cell, NK cell, or phagocyte (e.g., a monocyte, macrophage, ordendritic cell); (c) a second binding moiety that binds to a molecule onan immune system cell, e.g., a T cell, NK cell, or professionalphagocyte (e.g., a monocyte, macrophage, or dendritic cell); and (d) asortase recognition motif. In some embodiments the SRM is appropriatelypositioned to permit the agent to participate in a sortase-catalyzedreaction. In some aspects the disclosure provides a mammalian cell,e.g., a human cell, sortagged with such a trivalent agent. The firstbinding moiety may be any binding moiety that binds to a molecule on atarget cell. The second and third binding moieties may be the same ordifferent and may be any binding moieties that bind to a T cell, NKcell, or professional phagocyte. The trivalent agent may link thesortagged cell to two other cells and to a target cell. The sortaggedcell is linked to the target cell via the first binding moiety, and thetwo cells to which the second and third binding moieties bind are linkedto the target cell via the sortagged cell.

In general, mammalian cell(s) to be sortagged with the bispecific ortrivalent agent may be of any cell type or types in various embodiments.In some embodiments the cells comprise PBMCs, lymphocytes, NK cells,APCs, red blood cells, or platelets. The cells may be a purifiedpopulation or may be a mixed population. In some embodiments at least5%, 10%, 20%, 30%, 40%, or 50% of the cells are CD4+ T cells. In someembodiments no more than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or50% of the cells are cytotoxic cells. In some embodiments the cells arenot activated ex vivo to become effector cells. In some embodiments thecells are not genetically engineered. In some embodiments the cells aregenetically engineered, e.g., they may be CAR cells, may express arecombinant gene product comprising a TCR, cytokine, cytokine receptor,costimulator, or costimulator receptor, or agent that inhibits theeffect or production of immunosuppressive substances produced by tumors,pathogens, or Tregs. In certain embodiments the sortagged mammaliancells, in addition to being sortagged with the bispecific or trivalentagent, are also sortagged with one or more agents that inhibit theeffect or production of immunosuppressive substances produced by tumors,pathogens, or Tregs. Suitable agents are discussed herein.

In some embodiments mammalian cells sortagged with the bispecific agentor trivalent agent are administered to a subject, and the second bindingmoiety (and, optionally, the third binding moiety, if present) binds toan immune system cell in vivo. In general, an immune system cell towhich the second or third binding moiety binds may be an endogenousimmune system cell of a subject or may be an exogenous cell that hasbeen administered to a subject. If the immune system cell is anexogenous cell, the cell may in some embodiments have been sortaggedand/or genetically engineered as described herein. For example, the cellmay be a CAR cell, may produce a recombinant gene product comprising acytokine, cytokine receptor, costimulator, costimulator receptor,adhesion molecule, etc.

In general, the binding moieties may be of any type, e.g., proteins(e.g., antibodies, antibody fragments), nucleic acid aptamers, smallmolecules, etc. In some embodiments, the first binding moiety, secondmoiety, or both, comprises an antibody, antibody fragment, scFv, singledomain antibody, or any other moiety that comprises an antigen bindingdomain. In some embodiments the bispecific agent is a bispecificantibody. In some embodiments the bispecific antibody comprises twoscFv, wherein a first say binds to a molecule on a target cell, and thesecond scFv binds to a molecule on the surface of an immune system cell,e.g., a T cell, NK cell, or professional phagocyte. In some embodimentsthe bispecific antibody comprises two single domain antibodies (sdAb),wherein a first sdAb binds to a molecule on a target cell, and thesecond sdAb binds to a molecule on the surface of an immune system cell,e.g., a T cell, NK cell, or professional phagocyte. In some embodimentsthe bispecific antibody comprises an scFv and an sdAb, wherein eitherthe scFv or the sdAb binds to the target cell and the other binds to theimmune system cell. In some embodiments the two scFv, two sdAbs, or scFvand sdAb are attached to each other using click chemistry to form thebispecific agent, and the bispecific agent is attached to the cell usingsortase. In some embodiments, sortase is used to install click chemistryhandles on the two scFv, two sdAbs, or saFv and sdAb, and the clickchemistry handles are allowed to react, thus generating the bispecificagent. In some embodiments the two scFv, two sdAbs, or scFv and sdAb areproduced as a fusion protein to form the bispecific agent, and thebispecific agent is attached to the cell using sortase. In someembodiments a single polypeptide chain comprising two VH and two VLregions, optionally separated by spacer regions, is produced, and thebispecific agent is attached to the cell using sortase. A sortaserecognition motif may, be located at or near a C-terminus of the agent,which may be used to attach the agent to the cell. In certainembodiments the bispecific agent is a bispecific antibody, in someembodiments a bispecific antibody comprising two sdAbs, which may insome embodiments be attached to each other using click chemistry, andthen attached to the cell using sortase. In some embodiments, one arm ofthe bispecific agent recognizes an activating molecule on a T cell, suchas CD3 on T cells, and the other arm recognizes an antigen on a targetcell, such as a tumor cell. In some embodiments a bispecific agentcomprises a first scFv that binds to CD3 epsilon chain and a second scFvthat binds to CD19, EpCam, CD33, or any other tumor antigen. In someembodiments, for example, the bispecific antibody blinatumomab(anti-CD3/anti-CD19), MT103 (anti-CD3/anti-EpCam), or AMG330 may bemodified to comprise a sortase recognition motif and used to sortagmammalian cells, e.g., human immune system cells. In some embodiments anscFv from any of the afore-mentioned bispecific antibodies may becombined with a scFv or other binding moiety that binds to any tumorantigen. In some embodiments a humanized anti-CD3 scFv may be used,e.g., a humanized OKT3 scFv (see, e.g., Woodle E S, et al. J Immunol1992; 148:2756-63; Kipriyanov S M, et al. Protein Eng 1997; 10:445-53).In some embodiments a fully human anti-CD3 scFv may be used. In someembodiments a bispecific agent comprises two immune checkpointinhibitors, optionally that inhibit different immune checkpointproteins. In some embodiments a bispecific agent comprises an immunecheckpoint inhibitor and a targeting moiety. In some embodiments thetargeting moiety targets the cell to tumor cells or tumor vasculature.

In some embodiments cells are sortagged with or administered incombination with an agent that enhances immune system cell infiltration,e.g., T cell infiltration, into tumors. In some embodiments the agent isan anti-angiogenic agent, e.g., a VEGF receptor inhibitor such as anantibody or other binding moiety that binds to a VEGF receptor andinhibits it by, e.g., blocking binding of VEGF; an antibody or otherbinding moiety that binds to one or more members of the VEGF family; anagent comprising a soluble VEGF receptor extracellular domain (e.g.,VEGF-Trap). In some embodiments the agent enhances the expression ofICAM-1, ICAM-2, or VCAM-1 on endothelial cells. In some embodiments theagent binds to an endogenous molecule that may inhibit migration ofimmune system cells, e.g., lymphocytes, across the endothelial barrier.Examples of such molecules include endothelin B receptor. In someembodiments the agent is an endothelin B receptor inhibitor. In someembodiments the endothelin B receptor inhibitor is an antibody or otherbinding moiety that binds to the endothelin B receptor. In someembodiments the endothelin B receptor inhibitor is small molecule suchas BQ-788. In some embodiments the cells are immune system cells, whichin some embodiments may be sortagged with a second agent, wherein thesecond agent has anti-tumor activity, such as an immune checkpointinhibitor. In some embodiments the cells are red blood cells that aresortagged with an agent that has anti-tumor activity, such as an immunecheckpoint inhibitor. In some embodiments the cells are CAR cells.

In some aspects the invention provides compositions comprising any twoor more cells or cell populations described herein, wherein at least oneof the cells or cell populations comprises a non-genetically engineeredpolypeptide having an agent conjugated thereto by sortase. In someaspects the invention provides methods comprising using any two or morecells or cell populations described herein in the same method, e.g.,administering any two or more cells or cell populations described hereinto a subject, wherein at least one of the cells or cell populationscomprises a non-genetically engineered polypeptide having an agentconjugated thereto by sortase. All different combinations areenvisioned. In some embodiments the cells or cell populations are of thesame cell type, e.g., two or more red blood cells or red blood cellpopulations conjugated with different agents, or two or more T cells orT cell populations conjugated with different agent. In some embodimentsthe cells or cell populations are of different cell types, e.g., redblood cells and lymphocytes (e.g., T cells), In some embodiments the twoor more cells or cell populations are individually of use for the samepurpose, e.g., anti-tumor therapy. In some embodiments the two or morecells or cell populations may have an additive or synergistic effect. Insome embodiments a combination may comprise red blood cells comprising anon-genetically engineered polypeptide that is sortagged with a firstagent in combination with immune system cells, e.g., lymphocytes, PBMCs,NK cells, sortagged with the same agent or a different agent. Withoutlimiting the invention in any way, certain embodiments useful for cancertherapy may comprise (1) red blood cells that are sortagged with animmune checkpoint inhibitor or angiogenesis inhibitor in combinationwith immune system cells, e.g., T cells or PBMCs, sortagged with anagent comprising a binding moiety that binds to a tumor antigen; (2) redblood cells that are sortagged with an immune checkpoint inhibitor orangiogenesis inhibitor in combination with CAR cells that are sortaggedwith an agent that enhances immune system cell infiltration into tumors;(3) red blood cells that are sortagged with an immune checkpointinhibitor or angiogenesis inhibitor in combination with CAR cells thatcomprise a CAR that binds to a first tumor antigen and are sortaggedwith an agent that binds to a second tumor antigen.

A subject may be treated with various preparative regimens prior toadministration of sortagged cells. For example, lymphodepletion of thepatient before adoptive cell transfer, which eliminates T regulatorycells and other lymphocytes, is a component of many ACT regimens forcancer (Dudley M E, et al. Adoptive cell transfer therapy followingnon-myeloablative but lymphodepleting chemotherapy for the treatment ofpatients with refractory metastatic melanoma. J. Clin. Oncol. 2005;23:2346-2357). Such lymphocytes might otherwise compete with thetransferred cells for homeostatic cytokines such as interleukin-7 (IL-7)and IL-15. Lymphodepletion before ACT may use total body irradiation orcytotoxic drugs to deplete the lymphoid compartment of patients. Thetransferred T cells may be administered with appropriate growth factorsto stimulate their survival and expansion in vivo and/or such growthfactors may be administered separately to the patient prior to orfollowing administration of the cells. In some embodiments, moleculescapable of stimulating endogenous antigen presenting cells, such asToll-like receptor agonists may be administered. In some embodimentsboth T cells and APCs are administered.

In some embodiments cells may be sortagged with a detectable label sothat they can be detected in vivo or in a sample subsequently removedfrom a subject.

In some aspects, methods of modulating the immune system of a mammaliansubject are provided herein. In some embodiments a method of modulatingthe immune system comprises administering a living mammalian cell to amammalian subject, wherein a moiety comprising an immunomodulator,antigen, or epitope, is conjugated to a living mammalian cell usingsortase. In some embodiments a method of modulating the immune systemcomprises conjugating a moiety comprising an immunomodulator, antigen,or epitope to a living mammalian cell using sortase and administeringthe living mammalian cell to a mammalian subject.

In some embodiments, modulating the immune system comprises modulatingone or more biological activities of one or more types of immune systemcells. In some embodiments, modulating the immune system comprisesmodulating an immune response to an antigen. In some embodiments,modulating an immune response to an antigen comprises modulating one ormore biological activities of one or more types of immune system cellsexposed to the antigen. In some embodiments an immune response comprisesmigration, proliferation, or activation of one or more types of immunesystem cells. In some embodiments an immune response comprisesdevelopment of immature immune system cells into mature, functionalcells. In some embodiments an immune response comprises proliferationand/or activation of helper (CD4+) T cells specific for an antigen. Insome embodiments an immune response comprises proliferation and/oractivation of cytotoxic (CD8+) T lymphocytes (CTLs) specific for anantigen. In some embodiments an immune response to an antigen comprisesproduction of cytokines by, e.g., immune system cells specific for theantigen. In some embodiments an immune response comprises proliferationand/or activation of antibody-producing cells (plasma cells) and/orproduction of antibodies by such cells, wherein the antibodies bind toan antigen. In some embodiments an immune response comprises productionof memory T and/or B cells that are capable of providing a rapid immuneresponse to an antigen upon subsequent exposure to the antigen thatelicited their production. In some embodiments modulating an immuneresponse comprises modulating any one or more biological activities ofimmune system cells. In some embodiments modulating an immune responseto an antigen comprises modulating any one or more biological activitiesof immune system cells that are specific for the antigen. In someembodiments modulating an immune response to an antigen modulates animmune response to an entity comprising the antigen. For example,modulating an immune response to a pathogen-derived antigen modulatesthe immune response to a pathogen comprising the antigen or a cellexpressing the antigen or displaying the antigen at its surface. Theterm “pathogen-derived antigen” encompasses any antigen that isnaturally produced by and/or comprises a polypeptide or peptide that isnaturally genetically encoded by a pathogen, e.g., any of the variouspathogens mentioned herein. In some embodiments a pathogen-derivedantigen is a polypeptide, a polysaccharide, a carbohydrate, a lipid, anucleic acid, or combination thereof that is naturally produced by apathogen. In some embodiments a pathogen-derived antigen is naturallyencoded by a pathogen and is produced by an infected cell as a result ofthe introduction into the cell of the pathogen's genetic material thatencodes the antigen. In some embodiments a pathogen-derived antigen isat least partly exposed at the surface of a cell membrane, cell wall, orcapsule. In some embodiments a pathogen-derived antigen is a secretedvirulence factor of a pathogen. In some embodiments a pathogen-derivedantigen is an antigen that plays a role in entry of the pathogen into ahost cell. For example, the antigen may bind to a cell surface moleculeof a cell to be infected. In some embodiments a pathogen-derived antigenis a toxin. In some embodiments a pathogen may be an agent that rarelyif ever causes disease in healthy, immunocompetent individuals, but thatcauses disease in at least some individuals who are susceptible, e.g.,individuals who are immunocompromised.

In some embodiments, modulating an immune response comprises stimulating(enhancing, augmenting, eliciting) an immune response. In someembodiments “stimulating” an immune response encompasses causingdevelopment of an immune response, enhancing the capacity of a subjectto mount an immune response, or increasing an immune response in asubject who is currently mounting an immune response. In someembodiments enhancing the capacity of a subject to mount an immuneresponse results in a faster or more robust immune response. In someembodiments an immune response is directed towards foreign entities(e.g., pathogens), infected cells, cancer cells, or other undesirable(e.g., deleterious) cells or substances that comprise the antigen.

In general, a targeting moiety may comprise any of a variety ofdifferent moieties, which may be obtained using any suitable method. Insome embodiments a targeting moiety comprises an antibody, an antibodychain, an antibody fragment, an scFv, a VHH domain, a single-domainantibody, protein, or an aptamer, wherein the antibody, antibody chain,antibody fragment, scFv, VHH domain, single-domain antibody, protein, oraptamer binds to the target.

In some embodiments, methods disclosed herein of modulating an immuneresponse enhance an adaptive immune response against a pathogen,infected cell, tumor cell, or other undesired cell or substance. In someembodiments, methods disclosed herein of modulating an immune responseenhance an innate immune response against a pathogen, infected cell,tumor cell, or other undesired cell or substance. In some embodiments,methods disclosed herein of modulating an immune response enhance bothan adaptive immune response and an innate immune response. In someembodiments, methods disclosed herein enhance a T cell-mediated immuneresponse, e.g., against a pathogen such as a virus (e.g., HIV),bacterium (e.g., Mycobacterium), fungus (e.g., Aspergillus) or parasite(e.g., Plasmodium), or against a tumor cell or other undesired cell. Insome embodiments, methods disclosed herein enhance cell-mediatedcytotoxicity towards a pathogen, infected cell, or tumor cell. Forexample, in some embodiments methods disclosed herein enhance activityof CD8+ cytotoxic T cells against a pathogen, infected cell, or tumorcell.

In some embodiments a composition comprises sortagged mammalian cells,wherein the cells are sortagged with any moiety of interest. In someembodiments a composition comprises mammalian cells, sortase, and asortase substrate. In some embodiments a composition comprises up toabout 10¹⁴ cells, e.g., about 10, 10², 10³, 10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶,10⁷, 5×10⁷, 10⁸, 5×10⁸, 10⁹, 5×10⁹, 10¹⁰, 5×10¹⁰, 10¹¹, 5×10¹¹, 10¹²,5×10¹², 10¹³, 5×10¹³, or 10¹⁴ cells. In some embodiments the number ofcells may range between any two of the afore-mentioned numbers. In someembodiments a composition further comprises a growth factor, cytokine,adjuvant or costimulator. In some embodiments the one or more growthfactors or cytokines promotes maturation, survival, proliferation, oractivation of at least some of the cells. In some embodiments a cytokineis IL-2. In some embodiments a cytokine is IL-7, IL-12, IL-15, or IL-21.In some embodiments a cytokine is TNF-alpha. In some embodiments acomposition in which immune system cells are cultured or maintainedcomprises an antibody or ligand of a T cell receptor or portion thereof,e.g., an antibody to CD3. In some embodiments a composition in whichimmune system cells are cultured or maintained comprises one or moreadjuvants. In some embodiments a composition comprising immune systemcells comprises one or more adjuvants that induces expression of acostimulator by at least some of the immune system cells. In someembodiments the one or more adjuvants comprises a TLR ligand, PAMP orPAMP mimic, CD40 ligand, or anti-CD40 antibody. In some embodiments acomposition in which immune system cells are cultured or maintainedcomprises one or more costimulators. In some embodiments a costimulatoris expressed at the surface of APCs, e.g., DCs. In some embodiments acostimulator is soluble. In some embodiments a costimulator is attachedto a surface, e.g., a particle.

In some embodiments an immune response comprises maturation,proliferation and/or activation of lymphocytes, e.g., CD4+ helper Tcells, that are specific for the antigen, i.e., that express receptors(TCR, BCR) that bind to the antigen, e.g., with high affinity. In someembodiments, cell activation results in increased expression of one ormore cytokine genes.

Cells, e.g., sortagged mammalian cells, may be characterized orassessed, e.g., to determine whether they have one or more properties ofinterest, to determine the effect of sortagging, and/or to determine theeffect of the cells when administered to a subject. In some embodiments,cells may be assessed for secretion of one or more cytokines, presenceof a cell surface marker profile characteristic of an activated state,and/or possession of one or more functional activities such as cytotoxicactivity. In some embodiments effector function of lymphocytes (e.g., Tcells) may be demonstrated by IFNγ secretion after co-culture withtarget cells (e.g., tumor cells, infected cells, or cells that have beenloaded with or caused to express a target antigen). In some embodimentseffector function of lymphocytes may be demonstrated by increasedexpression of CD107a (LAMP-1), which may serve as a functional markerfor the identification of CD8+ T cell and natural killer cell activity(e.g., degranulation). Cytokine secretion may be assessed using, e.g.,ELISA assay, cytokine antibody arrays, etc. In some embodiments,presence or proliferation of T cells with specificity for a particularantigen may be assessed using peptide-MHC multimers (e.g., dimers,tetramers, pentamers, etc.) which can be used to identify or isolate Tcells specific for the peptide, or in some embodiments using CD1-lipidmultimers, which can be used to identify or isolate natural killer Tcells specific for the lipid. Methods for generating peptide-MHC orCD1-lipid multimers are well known in the art.

A wide variety of assays are available to assess cell viability and/orproliferation. For example, a cell membrane integrity assay (e.g.,ability to exclude a compound that is generally excluded from viablecells, such as trypan blue or 7-amino actinomycin D (7-AAD), cellularATP-based assay, a mitochondrial reductase activity assay, calceinstaining, a DNA content assay using a nucleic acid dye, a cellularmetabolism assay such as resazurin (sometimes known as AlamarBlue,etc.), MTT, XTT, and CellTitre Glo, etc., a protein content assay suchas SRB (sulforhodamine B) assay; nuclear fragmentation assays;cytoplasmic histone associated DNA fragmentation assay; PARP cleavageassay; TUNEL staining; annexin staining, CyQUANT® cell proliferationassays (Life Technologies). In some embodiments cytotoxicity may beassessed using a flow cytometry based assay, wherein a fluorochrome orother detectable label is used to stain non-viable or viable cells in acell population, and the cells are subjected to flow cytometry andquantified.

In some embodiments cytotoxicity (e.g., cytotoxic effect of sortaggedmammalian cells) may be assessed, if desired, using any suitable assay.In some embodiments a chromium-51 (⁵¹Cr) release assay may be used. In a⁵¹Cr assay, target cells are loaded with ⁵¹Cr and maintained underconditions in which cytolysis may occur. The label may then be releasedfrom the target cells by cytolysis. The label can be isolated bycentrifuging the samples and collecting the supernatants. Supernatantsfrom centrifugation can either be counted directly in a gamma counter,or mixed with scintillation fluid or dried on a substrate comprising asolid scintillant such as a LumaPlate™ and counted in a scintillationcounter. Cytotoxicity assays utilizing similar principles to thechromium-51 release assay (loading cells with a compound or compoundprecursor and detecting compound subsequently released from lysed cells)may be used, such as the DELFIA® cytotoxicity assay (Perkin Elmer). TheDELFIA is based on loading cells with an acetoxymethyl ester of afluorescence enhancing ligand. After the ligand has penetrated the cellmembrane the ester bonds are hydrolyzed within the cell to form ahydrophilic ligand, which no longer passes through the membrane. Aftercytolysis the released ligand is introduced to a europium solution toform a fluorescent chelate. The measured signal correlates directly withthe amount of lysed cells. In some embodiments a cytotoxicity assay isused to measure cell-mediated cytotoxicity, e.g., cytotoxic activity ofT cells or NK cells. In some embodiments the ability of cells to lysetarget cells expressing particular peptides or other antigens may betested using cells that have been loaded with or caused to express suchpeptides or antigens as target cells. In some embodiments a cytotoxiccell is characterized in that it produces perforin, granzyme(s) (e.g.,granzyme A, granzyme B, granzyme 3/K), and/or granulysin. In someembodiments production of such enzymes may be detected by flowcytometry. In some embodiments, cytotoxicity may be observed using,e.g., time lapse microscopy.

In some embodiments pro-apoptotic activity (e.g., pro-apoptotic effectof sortagged mammalian cells) may be assessed, if desired, using anysuitable assay. In some embodiments a TUNEL assay, DNA fragmentationassay, Annexin V assay, caspase assay, mitochondrial membrane potentialassay, etc.

The ability of administered sortagged mammalian cells to produce auseful therapeutic effect may be assessed using standard methods forassessing the effect of therapies in the particular disease that thecells are intended to treat. For example, anti-tumor effect may beassessed in a variety of non-human animal tumor models, e.g., xenograftmodels, non-human animals with tumors that arise spontaneously or as aresult of genetic engineering, etc. In some embodiments tumor(s) may beremoved from the body (e.g., at necropsy) and assessed (e.g., tumors maybe counted, weighed, and/or size (e.g., dimensions) measured). In someembodiments the size and/or number of tumors may be determinednon-invasively. For example, in certain tumor models, tumor cells thatare fluorescently labeled (e.g., by expressing a fluorescent proteinsuch as GFP) can be monitored by various tumor-imaging techniques orinstruments, e.g., non-invasive fluorescence methods such as two-photonmicroscopy. The size of a tumor implanted or developing subcutaneouslycan be monitored and measured underneath the skin. Any of a wide varietyof methods and/or devices known in the art may be used to assess tumorsin vivo in animals or in human subjects. Tumor number, size, growthrate, or metastasis may, for example, be assessed using various imagingmodalities, e.g., 1,2, or 3-dimensional imaging (e.g., using X-ray, CTscan, ultrasound, or magnetic resonance imaging, etc.) and/or functionalimaging (e.g., PET scan) may be used to detect or assess lesions (localor metastatic), e.g., to measure anatomical tumor burden, detect newlesions (e.g., metastases), etc. In human subjects, objective criteriasuch as the original or revised Response Evaluation Criteria In SolidTumors (RECIST) (Therasse P, et al. J Natl Cancer Inst (2000) 92:205-16;Eisenhauer, E., et al., Eur J Cancer. (2009) 45(2):228-47) or in thecase of lymphomas or leukemias, response criteria described in Cheson BD, et al. J Clin Oncol 2007; 10:579-86, may be used. As will beappreciated, in a clinical response may not be evident until a number ofweeks or months after therapy. For example, in the case of a response toimmune checkpoint inhibitor therapy, a regression in size of lesions maybe slower (e.g., delayed by about 6 months after initiation of therapy)as compared with the timing that is typically seen in the case ofresponses to standard chemotherapeutic agents. Therapeutic effectagainst a pathogen may, e.g., be assessed based on symptoms of infectionin pathogen-exposed animals or human subjects, and/or by detecting areduction in the presence of pathogens or pathogen-infected cells inbody fluids (e.g., blood), tissues, or organs in infected subjects thatreceive the therapy as compared with controls.

Animal models exist for a variety of different autoimmune diseases. Forexample, the Collagen Induced Arthritis (CIA) model is a commonly usedmodel that shares immunological and pathological similarities to humanrheumatoid arthritis. Arthritis is initiated by intradermal injectionsof Collagen Type II (CII) emulsified in Complete Freund's Adjuvant(CFA), e.g., in rodents, which causes an immune response generatingantibodies to CII. There is both a T cell and B cell component to thepathology. Experimental autoimmune encephalomyelitis is an inflammatorydemyelinating disease of the central nervous system (CNS) and is used asan animal model of human CNS demyelinating diseases, including multiplesclerosis and acute disseminated encephalomyelitis. EAE serves as aprototype for T-cell-mediated autoimmune disease in general. EAE can beinduced in a number of species, including mice, rats, guinea pigs,rabbits and primates. Commonly used antigens in rodents are spinal cordhomogenate (SCH), purified myelin, myelin protein such as MBP, PLP andMOG, or peptides of these proteins. It may also be induced by thepassive transfer of T cells specifically reactive to these myelinantigens. Therapeutic efficacy may be assessed based on, e.g., clinicalsymptoms and signs, histopathology (e.g., lesions, tissue destruction),presence of self-reactive T cells, etc. Animal models of type I diabetesinclude, for example, non-obese diabetic (NOD), BDC2.5 transgenic, andhumanized mice such as NOD.β2mnull.HHD mice, which lack murine-derivedMHC I and instead transgenically express human HLA-A2.1 molecules.

Cells may be administered in an effective amount, by which is meant anamount sufficient to achieve a biological response or effect ofinterest, e.g., reducing one or more symptoms or manifestations of adisease or condition or modulating an immune response. In someembodiments a composition administered to a subject comprises up toabout 10¹⁴ cells, e.g., about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ cells, or any intervening number or range. Insome embodiments between about 10⁵ and about 10¹² cells areadministered. In some embodiments between about 10⁵-10⁸ cells and about10¹¹-10¹³ cells are administered. In some embodiments a subject receivesa single dose of cells. In some embodiments a subject receives multipledoses of cells, e.g., between 2 and 5, 10, 20, or more doses, over acourse of treatment. In some embodiments a dose or total cell number maybe expressed as cells/m² or cells/kg. For example, a dose may be about10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ cells/m² or cells/kg anyintervening number of range. In some embodiments a course of treatmentlasts for about 1-2 months, 2-6 months, 6-12 months, or more, e.g.,indefinitely or until the subject is no longer in need of treatment. Insome embodiments a subject may be treated about every 2-6 weeks. One ofordinary skill in the art will appreciate that the number of cells,doses, and/or dosing interval may be selected based on various factorssuch as the weight, surface area, and/or blood volume of the subject,the condition being treated, response of the subject, etc. The exactnumber of cells required may vary from subject to subject, depending onfactors such as the species, age, weight, sex, and general condition ofthe subject, the severity of the disease or disorder, the particularcell(s), the identity and activity of agent(s) conjugated to the cells,mode of administration, concurrent therapies, and the like. It will beunderstood that the amount may be decided by the attending physicianwithin the scope of sound medical judgment. In some embodiments bothsortagged and non-sortagged cells may be administered.

In some embodiments one or more compounds is also administered once ormore to the subject in addition to administering cells. In someembodiments a compound is administered at least once prior to and/or atleast once after administration of the cells. In some embodiments acytokine is administered, wherein the cytokine is capable of enhancingsurvival, proliferation, maturation, activation, or activity of immunesystem cells. In some embodiments the cytokine is IL-2. In someembodiments the cytokine is IL-7, IL-12, IL-15, or IL-21. In someembodiments an adjuvant is administered. In some embodiments theadjuvant is capable of inducing APCs to express a costimulator. In someembodiments the adjuvant and/or cytokine is administered in the samecomposition as the cells. In some embodiments the adjuvant, cytokine,and/or cells are administered in different compositions.

In general, cells may be administered using any suitable route ofadministration. In some embodiments cells are administered to thecirculatory system, e.g., by infusion. In some embodiments cells areadministered intravenously. In some embodiments cells are administeredto or in the vicinity of a tumor or a site that may harbor tumor cells(e.g., a site from which a tumor was removed or rendered undetectable bytreatment or to which a tumor is prone to metastasize), site ofinfection, or site of potential infection (e.g., a break in the skinsuch as a wound, indwelling device, surgical site, etc.), or any site atwhich an effect, e.g., a therapeutic effect, is desired. In someembodiments cells are administered into or in the vicinity of an organthat is affected by a condition for which the cells have a therapeuticeffect. In some embodiments the organ is one in which a tumor is presentor from which a tumor has been removed or to which a tumor is prone tometastasize. In some embodiments the tumor is a primary tumor. In someembodiments the tumor is a metastatic tumor. One of ordinary skill inthe art will be aware that certain tumor types are prone to metastasizeto particular organs, i.e., they metastasize to those organs commonly orat least more frequently than to many or most other organs. For example,breast tumors are prone to metastasize to bone, liver, lung and brain;colorectal cancers are prone to metastasize to the liver and lungs. Insome embodiments the organ is the first, second, third, or fourth mostcommon organto which the particular tumor type that the individual hasmetastasizes. In some embodiments the tumor is a metastasis. In someembodiments cells are administered into the portal vein or hepaticartery to treat a liver condition, e.g., a liver cancer (e.g.,hepatocellular carcinoma) or liver infection. In some embodiments cellsare administered into the pancreatic artery to treat a conditionaffecting the pancreas, e.g., pancreatic cancer. In some embodimentscells are administered into the peritoneal cavity to treat a conditionaffecting the peritoneum, such as a tumor. Primary peritoneal cancer isa cancer of the cells lining the peritoneum and is a form ofmesothelioma. The peritoneal cavity is a common site of ovarian cancerspread or recurrence. Peritoneal metastases may arise from a variety ofprimary cancer, such as gastrointestinal cancers. In some embodimentscells are administered into the pleural space or thoracic cavity totreat a condition affecting the lungs or pleura, such as a lung canceror pleural mesothelioma. In some embodiments cells are administered intothe spinal canal to treat a condition affecting the brain or meningessuch as a tumor. In some embodiments cells are administeredintraocularly to treat a condition affecting the eye, such as a tumor.In some embodiments cells are administered into or in the vicinity of anorgan in which an infection has been detected or suspected of beingpresent Presence of an infection in an organ may be suspected e.g.,based at least in part on symptoms or signs experienced or exhibited bythe subject, detection of the infectious agent or a component thereof(such as DNA, RNA, protein) in a sample obtained from the organ, orknown propensity of the infectious agent to infect organs of that type.In some embodiments, “in the vicinity” of a site or organ refers towithin a location outside the organ or site and within 1 cm, 2 cm, 3 cm,4 cm, 5 cm from the edge of the organ or site. In some embodiments, “inthe vicinity” of a site or organ refers to administration into a bloodvessel that supplies the organ or site, either within the organ or siteor at a location no more 5 cm, 10 cm, 15 cm, 20 cm, or 25 cm away fromthe point where the blood vessel (or one or more blood vessels thatarise from the blood vessel) enter the organ or site. In certainembodiments cells may be introduced into a vessel that transports bloodout of or away from the organ. In some embodiments, such administrationmay be useful to target tumor cells that arise from a tumor within theorgan and enter the circulatory or lymphatic system.

In some embodiments, focused ultrasound (FUS) in the presence of amicrobubble contrast agent may be used to deliver sortagged cells to thebrain, e.g., for treatment of a tumor or infection in the brain usingimmune system cells targeted to an antigen expressed in the tumor, byinfected cells, or by an infectious agent. The FUS disrupts the bloodbrain barrier (BBB), facilitating delivery of cells to the brain. Insome embodiments the immune system cells are sortagged CAR cells. Insome embodiments the cells are sortagged with a targeting moiety.

Cells may be administered in any physiologically acceptable vehicle. Avehicle compatible with cell viability and not causing adverse effectswhen administered to a subject may be selected by the ordinary skilledartisan. In some embodiments a vehicle comprises water, appropriate saltconcentration, and may comprise a physiologically compatible buffersubstance such as HEPES.

In some embodiments immune system cells are administered to a subject inneed of prophylaxis or in need of treatment of an existing cancer or inneed of delaying, inhibiting, or preventing recurrence of cancer. Insome embodiments at least some of the introduced cells (or theirdescendants) mount an immune response against the cancer or againstcancer cells remaining in or arising in the body, wherein the cancer orcancer cells comprise the tumor antigen. In some embodiments at leastsome of the introduced cells (or their descendants) stimulatematuration, proliferation, and/or activation of at least some endogenousimmune system cells of the subject, e.g., endogenous T cells, whereinthe endogenous immune system cells mount an immune response against thecancer or against cancer cells remaining in or arising in the body,wherein the cancer or cancer cells comprise the tumor antigen.

In some embodiments a method comprises identifying an antigen expressedby a tumor for which a subject is in need of treatment. The tumor orcells obtained from the tumor can be analyzed for expression of tumorantigens using standard methods such as immunohistochemistry, flowcytometry, etc. In some embodiments, immune system cells are sortaggedwith an agent comprising a targeting moiety that binds to the antigen.The immune system cells and/or descendants thereof are subsequentlyadministered to the subject. In some embodiments immune system cells areobtained from a subject prior to treatment of the subject withchemotherapy or radiation. At least some of the immune system cells maybe stored for future use in producing one or more cell preparations tobe administered to the subject.

In some embodiments a subject, e.g., a subject to whom sortase-modifiedcells are administered, is immunocompetent, e.g., the subject has anormally functioning immune system. In some embodiments a subject isimmunocompromised. A subject may be immunocompromised for any of avariety of reasons. Such reasons may include, e.g., age (e.g., infantsor elderly individuals), genetic immunodeficiency disorders affectingone or more components of the innate and/or adaptive immune system,diseases such as cancer or infections that affect the immune system suchas HIV infection, treatment with an immunsuppressive or cytotoxic drug,e.g., for cancer (e.g., cancer chemotherapy) or to prevent or inhibittransplant rejection or to treat an autoimmune disease. Immunosuppresiveagents include, e.g., cytotoxic or cytostatic drugs, such as a varietyof chemotherapeutic drugs used in the treatment of cancer, various drugsadministered to reduce the likelihood of transplant rejection or totreat autoimmune diseases. Examples include, e.g., glucocorticoids,immunophilin-interacting agents such as rapamycin or rapamycin analogs,TNF alpha antagonists, etc.). In some embodiments a subject is atincreased risk of infection as compared with a normal, average healthyindividual, due, e.g., to hospitalization, surgery, chronic disease(e.g., diabetes, cancer, chronic obstructive pulmonary disease, cysticfibrosis), indwelling medical device (e.g., catheter, IV line), implantor prosthesis (e.g., heart valve replacement), physical trauma, burn,malnourishment, etc. In some embodiments sortase-modified cells are usedto induce or augment an immune response in a subject who has undergone,is undergoing, or will undergo chemotherapy or radiation therapy. Insome embodiments a subject is at increased risk of infection because thesubject is less than about 1 year of age or is over about 60, 65, 70,75, or 80 years of age.

In some embodiments, modulating an immune response comprises inhibitingthe immune response. As used herein, “inhibiting” an immune responseencompasses preventing or delaying development of an immune response toan antigen in a subject not currently exhibiting such response orreducing the intensity of a current or potential future immune response.In some embodiments an immune response is an unwanted immune response,e.g., an immune response that is deleterious to the subject in whom itoccurs. In some embodiments, an unwanted immune response is directedagainst self tissues or cells, transplanted tissue or cells, non-livingmaterials introduced into the body for diagnostic or therapeuticpurposes, or an allergen.

In some embodiments an unwanted immune response is an immune responsethat is excessive or inappropriately prolonged, such that it isdeleterious to the subject. For example, an immune response directedagainst an antigen derived from a pathogen that has infected a subjectmay initially be beneficial in terms of controlling the pathogen but maybe too intense or prolonged, such that it causes tissue damage to thesubject (e.g., cell-mediated or antibody-mediated tissue damage) orsymptoms due to excessive cytokine release.

An unwanted immune response may be mounted by a subject against atransplanted tissue or organs or cells, such as blood cells, stem cells,blood vessel, bone marrow, solid organ (e.g., heart, lung, kidney,liver, pancreas), skin, intestine, or cells derived from any of theforegoing. For example, pancreatic tissue, e.g., pancreatic islets, orisolated pancreatic beta cells, may be transplanted from a donor into asubject in need of treatment of diabetes, e.g., type I diabetes. In someembodiments a transplant (also termed a “graft”) comprises allogeneiccells or tissues (i.e., the donor and recipient are differentindividuals from the same species). In some embodiments a transplantcomprises xenogeneic cells or tissues (i.e., the donor and recipient areof different species). The immune response may be directed, e.g.,against one or more donor antigens, e.g., histocompatibility proteins(e.g., major or minor histocompatibility proteins) of the donor. Animmune response directed against a graft may be referred to as“rejection”. Rejection may result in damage to the graft, which mayreduce its function, may lead to graft failure, and may ultimatelyrequire removal of the graft. In some embodiments sortagged mammaliancells comprise regulatory T cells conjugated with a moiety that targetsthem to cells, tissues, or organs at risk of or exhibiting evidence ofrejection, e.g., acute graft rejection.

An unwanted immune response may comprise graft-versus-host disease(GvHD). GvHD may occur, for example, after an allogeneic stem celltransplant. In GvHD, grafted donor immune cells or their descendantsrecognize the recipient (e.g., recipient's cells) as foreign and mountan immune response thereto, e.g., a T cell-mediated immune response.Allogeneic hematopoietic stem cell transplantation can be a curativetreatment in a variety of hematopoietic disorders, immunodeficiencies,and leukemias and finds use in treatment of a variety of cancers whenchemotherapy has ablated the patient's immune system. GvHD can be alife-threatening complication of such transplants. In some embodimentssortagged mammalian cells may be used to treat, e.g., prophylactically,GvHD. In some embodiments sortagged mammalian cells comprise regulatoryT cells conjugated with a moiety that targets them to cells, tissues, ororgans at risk of or exhibiting evidence of GvHD. In some embodiments,regulatory T cells may be modified using sortase, e.g., with a targetingmoiety that targets them to graft-derived immune cells and/or to sitesat which such immune cells are active. Mesenchymal stem cells have beenreported to have a variety of beneficial effects in treatment ofgraft-versus host disease (GvHD). For example, they have been reportedto reduce proliferation of graft derived T-cells, inhibit alloreactiveT-cell responses and support hematopoietic stem cell (HSC) engraftment.In some embodiments, MSCs may be modified using sortase, e.g., with atargeting moiety that targets them to graft-derived T cells and/or tosites at which such T cells are active. In some embodiments MSCs may beconjugated using sortase with a moiety that targets them to cells,tissues, or organs at risk of or exhibiting evidence of GvHD. In someembodiments the regulatory T cells and/or MSCs are targeted to the skin,liver, or gastrointestinal tract. In some embodiments the regulatory Tcells and/or MSCs may be conjugated with an agent, e.g., a cytokine,that exerts an inhibitory effect on donor immune system cells involvedin GvHD. In some embodiments the regulatory T cells and/or MSCs may bederived from the same donor as the original grafted cells.

In some embodiments an unwanted immune response comprises an immuneresponse to an autoantigen (also referred to as a self antigen), e.g.,in a subject suffering from an autoimmune disease. Such inappropriateimmune responses may involve self-reactive T cells, autoantibodies, orboth. One of ordinary skill in the art will be aware of variousautoantigens involved in particular autoimmune diseases. Autoimmunediseases include, for example, acute disseminated encephalomyelitis,alopecia areata, antiphospholipid syndrome, autoimmune hepatitis,autoimmune myocarditis, autoimmune pancreatitis, autoimmunepolyendocrine syndromes, autoimmune uveitis, inflammatory bowel disease(Crohn's disease, ulcerative colitis), type I diabetes mellitus (e.g.,juvenile onset diabetes), multiple sclerosis, scleroderma, ankylosingspondylitis, sarcoid, pemphigus vulgaris, pemphigoid, psoriasis,myasthenia gravis, systemic lupus erythemotasus, rheumatoid arthritis,juvenile arthritis, psoriatic arthritis, Behcet's syndrome, Reiter'sdisease, Berger's disease, dermatomyositis, polymyositis, antineutrophilcytoplasmic antibody-associated vasculitides (e.g., granulomatosis withpolyangiitis (also known as Wegener's granulomatosis), microscopicpolyangiitis, and Churg-Strauss syndrome), scleroderma, Sjögren'ssyndrome, anti-glomerular basement membrane disease (includingGoodpasture's syndrome), dilated cardiomyopathy, primary biliarycirrhosis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease),transverse myelitis, and Guillane-Barre syndrome. Examples of certainautoantigens that are involved in some of these diseases are discussedbelow. In some embodiments sortagged mammalian cells comprise regulatoryT cells conjugated with a moiety that targets them to cells, tissues, ororgans at risk of or exhibiting evidence of damage in an autoimmunedisease.

In some embodiments, a method for inducing tolerance comprisesgenerating tolerogenic DCs, e.g., DCs that either delete autoreactive Tcells or induce regulatory T (Treg) cells, e.g., CD4+CD25-Foxp3+regulatory T cells. In some embodiments, a method results in reductionin the number and/or activity of Th17 cells. In some embodimentstolerogenic DCs are generated in vitro and administered to a subject. Insome embodiments tolerogenic DCs are generated by a method comprisesexposing DCs, e.g., immature DCs, in vitro, to an agent comprising (a) atargeting moiety that binds to a DC cell surface protein and (b) anantigen, wherein the antigen comprises a self-antigen or allergenicantigen. In some embodiments inhibiting the immune response e.g.,induction of tolerance or a tolerogenic state, is achieved by using asuitable concentration or amount of the agent and/or exposing cells orsubjects to appropriate cytokines. In some embodiments targeting anantigen to DCs in the absence of an effective amount of an adjuvantinhibits the immune response to the antigen that would otherwise occurand thereby results in increased tolerance to the antigen. In someembodiments a method of inhibiting an immune response comprisesadministering to a subject an agent comprising a targeting moiety thatbinds to DCs and an antigen, wherein the antigen comprises aself-antigen or allergenic antigen. In some embodiments the antigen isone to which the subject has previously exhibited or continues toexhibit or is at risk of exhibiting an unwanted, e.g., deleterious,immune response. In some embodiments the agent is administered withoutadministering an effective amount of an adjuvant. For example, the agentmay be administered in a composition that is substantially free ofadjuvants.

In some embodiments, a method for inducing or promoting tolerancecomprises generating modified mammalian Treg cells (“Tregs”) usingsortase and administering the cells to a subject. As known in the art,Tregs are capable of regulating, e.g., inhibiting, immune responses by,for example, suppressing effector T cells. In some embodiments Tregs aremodified by conjugating a targeting moiety to their surface usingsortase, wherein the targeting moiety targets the Tregs to an organ,tissue, or site in the body of a subject where regulation, e.g.,inhibition, of an immune response is desired. In some embodiments, theorgan, tissue, or site is a graft. In some embodiments the Tregs aregenerated by obtaining Tregs from a subject and modifying the Tregs exvivo using sortase. In some embodiments Tregs obtained from a subjectare expanded ex vivo prior to modification by sortase. In someembodiments modified Tregs may be contacted with a graft ex vivo priorto transplanting the graft into a receipient. The Tregs may infiltratethe graft and inhibit development or limit the extent of an immuneresponse after transplant. In some embodiments Tregs may be administeredtogether with the graft and/or after the transplant. Tregs may beadministered locally at or near the site of transplant and/or byintroducing the cells into the bloodstream or lymphatic system. In someembodiments at least one cytokine is administered to the subject one ormore times in combination with Tregs. A cytokine and Tregs may beadministered at the same site, different sites, or both. A cytokine maybe administered prior to, at the same time as, and/or afteradministration of the Tregs. If administered at the same time, the Tregsand cytokine(s) may be in the same composition as the Tregs or in adifferent composition. In some embodiments a cytokine is a cytokine thathas anti-inflammatory properties, e.g., IL-2, IL-10, transforming growthfactor-β (TGF-β), IL-27, IL-35 or IL-37. Such anti-inflammatoryproperties may comprise, e.g., inhibiting formation, maturation,expansion or activity of effector immune system cells; stimulatingformation, maturation, expansion or activity of regulatory T cells, etc.

Tregs may be isolated and, if desired, expanded ex vivo using methodsknown in the art (see, e.g., and references therein for examples of suchmethods). Tregs may be identified based on a cell surface markerexpression pattern of CD4+CD25+CD127lo. In some embodiments Tregs arecharacterized by high-level expression of CD25, FoxP3, CTLA-4, GITR, andCD62L and a very low or undetectable expression of CD127.

In some embodiments inhibiting an unwanted immune response comprisesstimulating an immune response against one or more cellular componentsof the unwanted immune response. For example, in some embodiments animmune response directed against self-reactive immune system cells,e.g., self-reactive T cells, is stimulated. In some embodiments animmune response directed against immune system cells at least in partresponsible for an immune-mediated disorder, e.g., allergy, isstimulated. In some embodiments an immune response directed against oneor more cellular components of the unwanted immune response at least inpart eliminates such cells, resulting in a reduction or inhibition ofthe unwanted immune response.

In some embodiments a composition, e.g., a composition to be used toinduce tolerance in a subject, is substantially free or essentially freeof any one or more substances, e.g., any one or more particularadjuvant(s), e.g., any one or more of the adjuvants or classes ofadjuvants mentioned above or known in the art. In some embodiments theconcentration or amount of adjuvant present, if any, is ineffective toenhance an immune response. In some embodiments the concentration oramount of adjuvant is less than or equal to 1%, 5%, 10%, 15%, 20%, or25% of the concentration or amount that would be effective to stimulatean immune response, e.g., an amount that would be used by one ofordinary skill in the art seeking to generate or enhance an immuneresponse against an antigen, e.g., in a vaccine. In some embodiments acomposition is substantially free or essentially free of any one or moreparticular adjuvant(s), e.g., any one or more of the adjuvants orclasses of adjuvants mentioned above or known in the art.

In some embodiments a method comprises identifying an antigen to which asubject is allergic or reactive (e.g., self-reactive) and conjugating anagent comprising the antigen to a mammalian cell using sortase. In someembodiments the cell is capable of inducing tolerance to the antigen. Insome embodiments the cell is a Treg cell. In some embodiments the cellis administered to a subject in need of treatment of an allergy or inneed of inhibition of immune response against the antigen. In someembodiments identifying an antigen comprises administering a test doseof one or more antigens to the subject, e.g., performing a skin test. Insome embodiments identifying comprises determining the response of thesubject to a test dose of one or more allergens or antigens. In someembodiments, if the response to an allergen is abnormally intense, theantigen is identified as one to which the subject is allergic orself-reactive. In some embodiments the subject harbors self-reactive Tcells or B cells comprising a TCR or BRC that recognizes the antigen. Insome embodiments the subject produces antibodies that bind to theantigen. In some embodiments a method comprises determining whether asubject produces antibodies that bind to an allergenic antigen orself-antigen. In some embodiments a sample comprising cells or serumfrom a subject is tested against a panel of candidate allergenicantigens or autoantigens in order, e.g., to identify one or moreallergenic antigens or self-antigens at least in part responsible forcausing an allergy or autoimmune disease.

In some embodiments cells are sortagged with an antigen to which thesubject is allergic or self-reactive. In some embodiments at least someof the cells are administered to the subject. In some embodiments thecells promote or induce tolerance to the antigen. For example, in someembodiments, a method for inducing or promoting tolerance comprisesgenerating modified mammalian cells using sortase, wherein the cells arenot genetically engineered for sortagging, and administering the cellsto a subject in need thereof, e.g., a subject suffering from or at riskof an autoimmune disease. The mammalian cells may be sortagged with anyautoantigen or allergen of interest or fragment thereof. In someembodiments the mammalian cells may be obtained from the subject or froman immunocompatible donor. In some embodiments the mammalian cellscomprise lymphocytes, PBMCs, splenocytes, or red blood cells. In someembodiments the antigen comprises a T cell epitope. In some embodimentsthe T cell epitope is a CD4+ epitope. In some embodiments the T cellepitope is a CD8+ epitope. Cells may be sortagged with an agentcomprising a single epitope or multiple epitopes, which may be from thesame or different antigens. In some embodiments multiple populations ofcells, sortagged with agents comprising different epitopes, may beadministered. In some embodiments a subject may be tested to identifyautoantibodies and determine which antigen(s) they react with. An agent(or agents) comprising one or more epitopes from those particularantigens may be conjugated to cells using sortase, for subsequentadministration to the subject. In some embodiments, inducing toleranceto an autoantigen may reduce the clinical severity (e.g., reduce theseverity of one or more symptoms), reduce the rate of progression,reduce the number of flare-ups, induce a remission, reduce the level ofone or more biomarkers or other indicators of the disease, or otherwiseshow evidence of beneficial effect.

In some embodiments an autoimmune disease to be treated is multiplesclerosis (MS). MS is an inflammatory demyelinating disease of the CNScharacterized by the formation of multiple discrete inflammatory lesionsand focal demyelination in perivascular and periventricular sites of CNSwhite matter (Nylander, A. & Hafler, D. A. (2012) J. Clin. Invest. 122,1180-1188). These demyelinating lesions are marked by infiltration ofactivated mononuclear cells and are associated with the appearance ofneurologic deficits. MS is believed to be an autoimmune disorder causedat least in part by T cells specific for immunodominant self-epitopes ofmyelin and other CNS antigens. It is thought that such autoreactive Tcells migrate into the CNS and undergo re-activation upon T cell antigenrecognition of endogenous CNS epitopes and secrete pro-inflammatorycytokines and chemokines which recruit inflammatory macrophages andother leukocytes from the blood to initiate focal demyelination and CNSdysfunction. In some embodiments the autoantigen is a myelin protein ofthe central nervous system (CNS) such as myelin basic protein (MBP),proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG).In some embodiments mammalian cells, e.g., RBCs or PBMCs, are sortaggedwith an agent comprising any of these proteins or an agent, e.g., apeptide, comprising an epitope found in any of these proteins, andadministered to a subject in need of treatment for MS. In someembodiments the peptide comprises MBP amino acids 89-99.

In some embodiments an autoimmune disease to be treated is type Idiabetes (T1D). In some embodiments cells, e.g., RBCs or PBMCs, aresortagged with an agent that comprises insulin (INS), islet-specificG6Pase catalytic subunit-related protein (IGRP), heat shock protein 60(HSP60), islet cell antigen 512 (IA-2), or other islet cell antigens, oran agent, e.g., a peptide, that comprises an epitope of any of theseproteins. For example, the antigen may comprise insulin B amino acids9-23. In some embodiments the cells may be administered to a subject inneed of treatment for T1D. In some embodiments the cells may beadministered to a subject with T1D who is to receive or has received atransplant comprising islet cells, e.g., isolated islet cells, islets,or pancreatic tissue comprising islets.

In some embodiments an autoimmune disease to be treated is an autoimmuneblistering skin disease, which term refers to a group of diseasescharacterized by autoantibodies against structural components of theskin. Further details regarding these diseases and particularautoantigens and epitopes implicated in their pathogenesis may be foundin Otten, J V, et al., Curr Mol Med. January 2014; 14(1): 69-95, andreferences cited therein. In pemphigus vulgaris (PV) autoantibodiesreact mainly with desmoglein 3 (Dsg3) alone and/or in combination withdesmoglein 1 (Dsg1). In bullous pemphigoid (BP) autoantibodiesfrequently target two hemidesmosomal proteins, BP180 (collagen XVII) andBP230, but may also target other proteins such as plectin and a6integrin. In mucous membrane pemphigoid autoantibodies target severalautoantigens of the dermal-epidermal junction, including BP180, BP230,laminin 332, α6β4 integrin, and collagen VII. Autoimmunity to collagenVII is typically associated with the skin blistering diseaseepidermolysis bullosa acquisita (EBA), but also occurs occasionally inpatients with systemic lupus erythematosus or inflammatory boweldisease. Anti-p200 pemphigoid is an autoimmune subepidermal blisteringdisease, characterized by autoantibodies against a 200-kDa protein(p200) of the epidermal basement membrane, which has been identified asthe laminin yl chain. In some embodiments cells, e.g., RBCs or PBMCs,are sortagged with an agent comprising Dsg1, Dsg3, BP180, BP230, lamininγ1, collagen VII, plectin, a6 integrin, laminin 332, or α6β4 integrin oran agent, e.g., a peptide, that comprises an epitope found in Dsg1,Dsg3, BP180, BP230, laminin γ1, collagen VII, plectin, α6 integrin,laminin 332, or α6β4 integrin. In some embodiments the epitope is in theectodomain of the relevant protein. The cells may be administered to asubject in need of treatment for the relevant autoimmune blistering skindisease, SLE, IBD, or other autoimmune disease.

In some embodiments an autoimmune disease to be treated is aninflammatory bowel disease, e.g., Crohn's disease and ulcerativecolitis. Autoantibodies against exocrine pancreas (PAb) have beenreported to be pathognomonic markers of Crohn's disease (CD). Forexample, the glycoproteins CUZD1 and GP2 are targets of PAb in patientwith Crohn's disease. In some embodiments cells, e.g., RBCs or PBMCs,are sortagged with an agent comprising CUZD1 or GP2 or an agent, e.g., apeptide, that comprises an epitope found in CUZD1 or GP2. The cells maybe administered to a subject in need of treatment for Crohn's disease.

In some embodiments an autoimmune disease to be treated is aninflammatory arthritis, e.g., rheumatoid arthritis (RA). Autoantibodiesto type II collagen (CII), heat shock proteins such as theimmunoglobulin binding protein (BiP), citrullinated peptides (anti-CCP)and other citrullinated proteins such as vimentin and fillagrin arefound in patients with RA In some embodiments cells, e.g., RBCs orPBMCs, are sortagged with an agent comprising type II collagen (CII),heat shock proteins immunoglobulin binding protein (BiP), humanchondrocyte glycoprotein 39, citrullinated peptides (anti-CCP) andcitrullinated proteins such as citrullinated fillagrin or citrullinatedvimentin or an agent, e.g., a peptide, that comprises an epitope foundin any of these. The cells may be administered to a subject in need oftreatment for an inflammatory arthritis, e.g., rheumatoid arthritis.

In some embodiments mammalian cells are sortagged with an agent that iscapable of inhibiting or reducing the effect of (neutralizing) a toxicsubstance that may be present in the body of a subject, e.g., in theblood. A toxic substance is a substance that causes or is capable ofcausing death, injury, damage, or other physiological disturbance toorganisms when a sufficient quantity is introduced into or onto orabsorbed by a living organism. Toxic substances include those substancesrecognized as such in the art. The action of a toxic substance may be bychemical reaction or other activity on the molecular scale. In someembodiments a toxic substance exerts its effects when present in theblood or when transported via the circulatory system to one or morelocations in the body. The agent may be, e.g., an antibody, at least aportion of a receptor that binds to the toxic substance, or any otherbinding agent that binds to the toxic substance.

As used herein, a “toxin” is a toxic substance produced within livingcells or organisms. In some embodiments a toxin may be produced by orgenetically encoded by a microbe, e.g., a pathogen and/or may beproduced in the body as a result of infection by a pathogen. Toxins maybe produced, e.g., by bacteria, fungi, plants, protozoa, and parasites.In some embodiments a toxin is an exotoxin, i.e., it is released, e.g.,secreted, by cell(s) that produce it. Examples of toxins include AB.toxins, e.g., AB₁ toxins, AB₅ toxins. As used herein, an “AB₁ toxin” isa toxin that comprises an A subunit and a B subunit. It will beunderstood that a subunit, e.g., an A subunit, may be cleaved to producetwo polypeptide chains, which may be linked by one or more disulfidebonds. Diphtheria toxin (C. diphtheriae) is an exemplary AB1 toxin.Heparin-binding epidermal growth factor-like growth factor serves as areceptor for diphtheria toxin. Pseudomonas exotoxin A, another bacterialAB₁ toxin, utilizes the low density lipoprotein receptor-relatedprotein, also known as the a2-macroglobulin receptor to enter cells. AB₁toxins also include certain type II ribosome inactivating plant toxinssuch as ricin, abrin, cinnanomin, viscumin, ebulin, and nigrin b(Hartley, M R & Lord, J M, Cytotoxic ribosome-inactivating lectins fromplants, Biochim Biophys Acta, 1701 (1-2): 1-14, 2004; Xu H, et al.,Cinnamomin˜a versatile type II ribosome-inactivating protein. ActaBiochim Biophys Sin (Shanghai) 36(3): 169-76). A complete AB₅ toxincomplex contains six protein units, five B subunits that are similar oridentical in structure and a single A subunit. The A subunit (or aportion thereof) of an AB₅ toxin is the portion of the complexresponsible for toxicity. The B subunits form a pentameric(five-membered) ring, into which the A subunit extends and is held. TheB subunits may protect the A subunit and mediate binding to cells.Examples of AB₅ toxins (and names of bacteria that produce them)include, e.g., Campylobacter jejuni enterotoxin (C. jejuni), choleratoxin (V. cholerae), heat-labile enterotoxins LT and LT-II (Escherichiacoli), pertussis toxin (B. pertussis), shiga toxin (S. dysenteriae),shiga-like toxin (also known verotoxin) SLT1 and SLT2 (certain E. coli).Other toxins of interest include, e.g., Botulinum neurotoxin (C.botulinum), tetanus neurotoxin (C. tetani), and the large clostridialtoxins known as Toxin A and Toxin B (C. difficile).

In some embodiments a toxic substance, e.g., a toxin, is a cytolysin,which term refers to substances, e.g., proteins and lipids, that causelysis of cells, e.g., by damaging their cell membrane. In someembodiments a toxin is a hemolysin, which term refers to substances thatcause lysis of red blood cells Hemolysins can be identified by theirability to lyse red blood cells in vitro. In some embodiments ahemolysin may, in addition to affecting red blood cells, also affectother cells, such as leukocytes. In some embodiments a toxin is apore-forming toxin (PFT). PFTs, which include certain cytolysins, can bedivided into the following subcategories: alpha-pore-forming toxins(e.g., cytolysin A of E. coli, aerolysin (Aeromonas hydrophila), andClostridial Epsilon-toxin), beta-pore-forming toxins (e.g., α-hemolysin,Panton-Valentine leukocidin, Vibrio cholerae cytolysin, and S. aureusgamma-hemolysin, Clostridium perfringens enterotoxin). Other cytolysinsinclude, e.g., anthrax toxin, cholesterol-dependent cytolysins such aspneumolysin, and small pore-forming toxins such as gramicidin A.

In certain embodiments a toxin is a protein, e.g., an enzyme, thatdegrades or directly damages host cell molecules or tissues. Examples,include, e.g., hyaluronidase, proteases, coagulases, lipases,deoxyribonucleases. In certain embodiments a toxic substance is asuperantigen or superantigen-like protein. Superantigens (SAgs) are aclass of antigens that cause non-specific activation of T-cellsresulting in polyclonal T cell activation and massive cytokine release.T cell activation is believed to result from SAg-mediated cross-linkingof major histocompatibility complex (MHC) class II antigens and T-cellreceptors (TCRs). SAgs contain distinct domains capable of binding toMHCII and TCR. SAgs can be produced by pathogenic microbes (e.g.,certain bacteria) or may be endogenous and produced in response toinfection by pathogenic microbes (e.g., certain viruses). Many SAgs areexotoxins, a number of which are produced by the Gram-positive organismsStaphylococcus aureus and Streptococcus pyogenes. SAgs are the causativeagents in toxic shock syndrome, among other disorders. Manysuperantigens share a common architecture that is also shared by thesuperantigen-like proteins (SSL), another group of bacterial virulencefactors. Certain Sags and SLLs are discussed in Fraser J D, Proft T. Thebacterial superantigen and superantigen-like proteins. Immunol Rev.2008; 225:226-43.

In some embodiments a toxin is an endotoxin, which refers to toxins thatare typically not released from the cells that produce them (except inthe case of destruction or damage to the cell, e.g., destruction of thebacterial cell wall). Examples of endotoxins are lipopolysaccharide(LPS) or lipooligosaccharide (LOS), found in the outer membrane ofvarious Gram-negative bacteria. The toxic effects of endotoxins onvertebrate organisms are mediated by their interaction with receptors onimmune system cells, which results in synthesis and/or release of immunemediators such as cytokines, nitric oxide, and eicosanoids, excessiveamounts of which can damage the organism.

Further information regarding certain toxins discussed above and manyothers may be found, e.g., in Alouf, J E & Popoff, M R, (eds.) TheComprehensive Sourcebook of Bacterial Protein Toxins, Third Edition,Academic Press, 2006; Schmitt, M J & Schaffrath, R (eds.) MicrobialProtein Toxins, Topics in Current Genetics 1 1, Berlin, N.Y.:Springer-Verlag, 2005; Pro ft, T. (ed.) Microbial toxins: molecular andcellular biology, Norfolk, England: BIOS Scientific, c2005.

In some embodiments a toxic substance may not be directly toxic but mayenhance the effect of a toxic substance or may be required for itsactivity. For example, the substance may mediate entry of a toxicsubstance into cells. In some embodiments a substance may be convertedinto a toxic substance in the body.

In some embodiments a subject may be accidentally or deliberatelyexposed to a toxic substance. In some embodiments a toxic substance maybe a pesticide (e.g., an insecticide or herbicide), a chemical used orproduced in industrial processes, etc. In some embodiments a toxicsubstance may be a therapeutic agent that has been administered to asubject in an excessive amount and/or has been administered to a subjectwho has reduced (e.g., below normal) capacity to metabolize or excretethe substance.

In some embodiments a toxic substance may be produced by a subject's owncells, e.g., in response to infection or as part of a disease process.For example, certain cytokines and inflammatory mediators produced inresponse to infection or injury or in certain diseases may have damagingeffects when present in excessive amounts. Examples include, e.g.,pro-inflammatory cytokines such as interferon gamma, TNF-alpha, IL-1(e.g., IL-1β), IL-6, and IL-17, and inflammatory mediators such asleukotrienes.

In some embodiments a toxic substance is a virulence factor or componentthereof. Virulence factors are substances or structures produced orencoded by pathogens (bacteria, viruses, fungi, protozoa, ormulticellular parasites) that play a role in establishing and/ormaintaining an infection. A virulence factor may permit or increase theability of a pathogen to achieve one or more of the following:colonization of a niche in the host, immunoevasion, evasion of thehost's immune response, immunosuppression, entry into and/or exit out ofcells or cellular compartments (if the pathogen is an intracellular oneduring at least part of its life cycle), obtain nutrition from the host.Virulence factors include, e.g., pathogen-produced toxins, adhesivemolecules (e.g., adhesins), molecules that stimulate endocytosis,immunoglobulin-binding proteins, proteases that degrade immunoglobulinsor other host cell molecules that play a role in the immune response,bacterial capsule, which may inhibit phagocytosis of the bacteria byhost immune cells, complement inactivating molecules, structures such aspili or fimbriae. In some embodiments a virulence factor is a biofilmcomponent. In some embodiments a virulence factor is encoded by aplasmid or bacteriophage.

Cells may be sortagged with any suitable moiety capable of binding toand/or inhibiting a toxic substance or virulence factor. In someembodiments an inhibitor of a toxic substance or virulence factor maybind to the substance or structure and thereby prevent it from acting onor interacting with its target, may inactivate the substance orstructure (e.g., by cleaving it), etc. It will be understood thatinhibition may be partial or complete. Examples of suitable inhibitingmoieties include, e.g., proteins, aptamers, or other moieties that arecapable of binding to the substance or structure, enzymes that arecapable of cleaving the substance or structure, etc. In some embodimentsan inhibitor of a toxic substance comprises a naturally occurringreceptor for the substance or a fragment or variant of the receptor,wherein the variant or fragment is sufficient for binding the substance.A variety of agents capable of inhibiting toxic substances are known inthe art and may be incorporated into agents that are used to sortagmammalian cells.

In some embodiments, cells may be sortagged with an agent capable ofbinding to a pathogen or pathogen-secreted molecule, e.g., any pathogenor pathogen-secreted molecule of interest. In some embodiments thepathogen is one that is typically present in the blood of a vertebrate,e.g., mammalian, host during all or part of the pathogen's life cycle.In some embodiments the pathogen-secreted molecule is secreted into theblood or gains entry into the blood of a vertebrate, e.g., mammalian,host infected by the pathogen.

In some embodiments living mammalian cells may be sortagged with adetectable label. The sortagged cells may be administered to a subjectand subsequently detected in vivo or in a sample obtained from thesubject. The detectable label may be selected to permit in vivodetection, e.g., by an imaging technique such as ultrasound, PET scan,MRI, fluorescence detection (e.g., near infrared). In some embodimentsliving mammalian cells may be sortagged with detectable label and atargeting moiety capable of targeting the cells to a target of interest,e.g., in the body of a subject. The cells become attached to the target,thereby concentrating the detectable moiety at the target. Detection ofthe detectable label may allow detection of the target. The target maybe any molecule, cell, or structure in the body of a subject. In someembodiments the target may be a pathogen, pathogen component, orpathogen-secreted substance. In some embodiments the target may be atoxic substance.

In some embodiments living mammalian cells sortagged with a detectablelabel may be administered to a subject together with other cells of thesame type that are not sortagged. The distribution and/or concentrationof sortagged cells may be representative of the distribution and/orconcentration of the administered population. Such sortagged cells mayserve as tracking agents, e.g., detection and/or quantification of thesortagged cells provides an indication of the distribution and/orconcentration of the administered population. For example, whenadministering cells for adoptive immunotherapy it may be of interest todetermine where such cells localize and/or their average residence timein the body. In some embodiments a population of cells may be isolatedbased on any one or more criteria or properties of interest, such as aparticular cell surface marker expression pattern, gene expressionprofile, functional activity, or based on their having been generatedthrough or subjected to a particular protocol or exposed to particularagents. Cells may be sortagged with a detectable label and mixed withone or more other cell populations (which may or may not be sortagged,e.g., with different detectable label(s) or other moietie) oradministered to a subject. The label(s) may be used to detect the cellin vitro or in vivo. For example, it may be of interest to monitor cellmigration, cell-cell physical interactions, cell division, or celldistribution.

In some embodiments, sortase-modified mammalian may be used inregenerative medicine. Regenerative medicine as used herein refers totherapies that comprise replacing or regenerating mammalian, e.g.,human, cells, tissues or organs to improve function, e.g., to restore orestablish normal function and/or structure, by administering cells tothe subject and/or by administering biologically active substances thatact on endogenous cells or tissues to promote their healing orregeneration. Examples of regenerative medicine therapies include usingimplanted cartilage cells (e.g., chondrocytes) to restore cartilage,strategies to remuscularize the injured heart, e.g., using adult stemcells, pluripotent stem cells, or cardiomyoctes, ex vivo production oftissues or organs which may then be implanted into subjects, amongothers.

Sortase-modified mammalian cells may be used in regeneration of any of awide variety of tissues and organs. Tissues and organs of interestinclude, e.g., cartilage, bone, heart, heart valve, blood vessel,esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum,anus, endocrine gland (e.g., thyroid, parathyroid, adrenal, endocrineportion of pancreas, e.g., islets of Langerhans), skin, hair follicle,tooth, gum, lip, nose, mouth, thymus, spleen, skeletal muscle, smoothmuscle, joint, brain, spinal cord, peripheral nerve, ovary, fallopiantube, uterus, vagina, mammary gland, testes, vas deferens, seminalvesicle, prostate, penis, pharynx, larynx, trachea, bronchi, lungs,kidney, ureter, bladder, urethra, eye (e.g., conjunctiva, retina,retinal pigment epithelium, cornea), or ear (e.g., organ of Corti). Insome embodiments, a tissue is an epithelial layer, e.g., an epitheliallayer lining the interior of a hollow organ. Regenerative medicineencompasses tissue engineering, i.e., the use of living cells seeded ona natural or synthetic extracellular substrate to create implantablestructures, e.g., parts of an organism.

In some embodiments sortase is used to conjugate a moiety to a cell thatis subsequently administered to a subject in need of regeneration, e.g.,of a tissue or organ. In some embodiments the cell may be administeredinto, adjacent to, or near (e.g., within 5 cm, 10 cm, 20 cm, or 25 cm) atissue or organ whose regeneration is desired. For example, if cardiacregeneration is desired, the cells may be administered to the heart. Insome embodiments the cell may be administered into the circulatorysystem. In some embodiments a moiety comprises a targeting moiety bindsto a target at a location in the body at which regeneration is desired.In some embodiments the target may be a cell-type specific markerexpressed by cells in the tissue or organ to be regenerated. Thetargeting moiety may enhance attachment of the administered cell to asite where regeneration is desired or may enhance integration of theadministered cell into a tissue or organ whose regeneration is desired.In some embodiments a moiety may promote survival, proliferation, orfunctional activity of the administered cell. In some embodiments themoiety may promote survival, proliferation, or functional activity ofcells found in a tissue or organ whose regeneration is desired. In someembodiments the moiety may promote migration of circulating or residentadult stem cells to a tissue or organ whose regeneration is desired ormay promote retention, functional integration, and/or differentiation ofsuch adult stem cells. Examples of moieties that may be useful forregenerative medicine include, e.g., growth factors, survival factors,cell adhesion molecules,

In some embodiments sortase-modified cells (e.g., conjugated with anagent that promotes cell survival, proliferation, functional activity,or tissue integration) may be used in the production of tissues ororgans ex vivo, which may then be implanted into a subject. For example,they may be used, optionally in combination with unmodified cells, toseed two-dimensional or three-dimensional scaffolds (sometimes termed“matrices” or “constructs”) ex vivo, or they may be used to provideappropriate stimulatory signals (e.g., growth or survival signals) tocells that are used in the production of tissues or organs ex vivo. Ascaffold promote cell-biomaterial interactions, cell adhesion, and ECMdeposition, may permit sufficient transport of gases, nutrients, andregulatory factors to allow cell survival, proliferation, anddifferentiation, and may in some embodiments biodegrade at a rate thatapproximates the rate of tissue regeneration under the cultureconditions or in vivo conditions of interest. In some embodiments ascaffold is comprised of materials that provoke no or minimal degree ofinflammation or toxicity in vivo. Scaffolds may comprise, e.g.,decellularized structures such as decellularized blood vessels ororgans, or may comprise synthetic scaffolds such as those produced usingvarious synthetic polymers. Polymer scaffolds may be porous and/orbiodegradable in certain embodiments. Examples of polymers or polymercompositions of natural origin of use in forming scaffolds includecollagen, gelatin, fibrin, hyaluronic acid, alginate, and chitosan.Synthetic polymers of use in forming scaffolds include polyglycolide,poly(L-lactic acid), poly(l-lactide-co-glycolide), poly(ε-caprolactone).It will be appreciated that derivatives, copolymers, and blends ofnatural and/or synthetic polymers may be used. Decellularization canprovide an acellular, three-dimensional biologic scaffold that can beseeded with selected cell populations. A scaffold may be composed of ECMproteins typically found in the body. The three-dimension architectureof a scaffold may be similar to that of the original tissue or organ,which may thus confer appropriate mechanical and physical properties.Agents conjugated to sortase-modified cells may, e.g, promote cellattachment to a scaffold, may promote cell-cell adhesion, may promotecell survival, proliferation, and/or differentiation.

VII. Kits and Services for Sortagging Mammalian Cells and/or OtherEukaryotic Cells

In some aspects, the invention provides kits useful for generatingsortagged mammalian cells, wherein the cells are not geneticallyengineered for sortagging. In some embodiment a kit comprises (i) asortase polypeptide, a nucleic acid or vector that encodes a sortasepolypeptide, or a cell line that expresses sortase polypeptide; and (ii)one or more items useful in the preparation, characterization, and/orpurification of sortagged mammalian cells. In s one or more items may beany of the items that are described herein with regard to methods ofpreparing, characterizing, and/or purifying sortagged living mammaliancells that are not genetically engineered for sortagging. In someembodiments the one or more additional items are selected from: (a) anagent comprising a transamidase recognition sequence; (b) mammaliancells that are not genetically engineered for sortagging; (c) a liquidcomposition, or components thereof, suitable for use as a reactionbuffer in which to sortag living mammalian cells; (d) one or morereagents useful for separating sortagged cells from sortase; and (e) acontrol substance. In some embodiments a kit comprises instructions forpreparing sortagged mammalian cells that are not genetically engineeredfor sortagging. In some embodiments instructions may be made availableseparately from the kit. For example, instructions may be provided oraccessed via the Internet, e.g., on the World Wide Web (“web”). In someembodiments the agent comprising a transamidase recognition sequencecomprises a binding moiety that binds to a tumor antigen. The agent maybe used, e.g., to sortag cells that are to be administered to a subjectin need of treatment for a tumor that expresses the TA. In someembodiments, any of the afore-mentioned kits may not comprise a sortase.For example, the agent comprising a transamidase recognition sequencemay be provided alone or together with one or more of theafore-mentioned additional items.

In some embodiments, a kit comprises a plurality of distinct agents eachcomprising a transamidase recognition sequence, wherein distinct agentseach comprise a different binding moiety. In some embodiments, each of aplurality of different binding moieties present in agents in the kitbinds to a different tumor antigen. A health care facility, e.g., ahospital, that treats patients in need of treatment for tumors mayobtain such a kit and may use it on site to sortag cells to beadministered to the patient. The particular agents to be conjugated tocells may be selected from those present in the kit, based at least inpart on results of analyzing expression of tumor antigens on aparticular patient's tumor. In some embodiments, a kit comprises atleast 5, 10, 15, 20, 25, 30, 40, or 50 different agents, e.g., up toabout 100, 200, 500, or more agents, each comprising a binding moietythat binds to a different TA. Any one or more of the TAs mentionedabove, in any combination, may be represented by agents in the kit. Insome embodiments sortase is provided in the kit, optionally togetherwith any one or more of the additional agents mentioned above. In someembodiments sortase may be provided separately.

In some aspects, the invention provides methods in which sortagging ofeukaryotic cells, e.g., mammalian cells, may be performed as a service.An organization or individual that performs sortagging for otherorganizations and/or individuals, e.g., upon request, may be referred toas a “sortagging service provider”. In some embodiments a sortaggingservice provider may be in the business of providing services, e.g.,research services, manufacturing services, to the pharmaceuticalindustry, biotechnology industry, biomedical research community, etc. Insome embodiments a sortagging service provider may have a website andmay offer sortagging services on its website. In some embodiments asortagging service provider receives a request (also referred to as an“order”) for sortagged mammalian cells from a requestor, which may beany organization or individual that seeks to obtain sortagged mammaliancells. An organization may be a for-profit organization, a non-profitorganization, a company, a corporation, a contract researchorganization, a research institution, an academic institution, etc. Arequest may be submitted, transmitted, received, and/or stored at leastin part electronically or in electronic format. In some embodiments arequest is submitted via a webpage and transmitted via the Internet. Arequest may indicate the type of cells and/or the agent(s) to beconjugated to the cells. In some embodiments a request may indicate oneor more additional specification such as the number of cells,manufacturing conditions (e.g., research grade, clinical grade (e.g.,GMP-compliant), storage conditions, shipping conditions, etc. In someembodiments the requestor may provide the cells and/or may provide oneor more agent(s) to be conjugated to the cells or one or more moietiesto be incorporated into agent(s) to be conjugated to cells. For example,in some embodiments a requestor may provide cells that originate fromand/or are designated for administration to a particular subject. Thesortagging service provider performs or arranges for sortaggingaccording to the request and supplies sortagged cells, e.g., to therequestor or as indicated by the requestor. A sortagging serviceprovider may perform the sortagging and/or providing itself and/orthrough arrangements with other organizations or individuals. In someembodiments a sortagging may be performed for a fee, under a contract,or according to a quote. A sortagging service provider may offer one ormore ancillary services that may facilitate or support or may be usefulin connection with generating or using sortagged mammalian cells.Examples include, e.g., cell expansion services, preparation of agentsto be conjugated to mammalian cells, characterization of sortagged cells(e.g., assays of cell function or biological activity of an agentconjugated to the cells). In some embodiments, a sortagging serviceprovider analyzes a tumor sample from a subject who is in need oftreatment for a tumor and identifies one or more tumor antigensexpressed by cells of the tumor, to which therapeutic cells are to betargeted. The sortagging service provider may sortag cells to beadministered to the subject with one or more targeting moieties thatbind to the tumor antigen(s). The sortagged cells may be transported toa location where they are administered to the subject.

In some embodiments, any of the above-mentioned kits and services may beused for the sortagging of non-mammalian eukaryotic cells, e.g., cellsof non-mammalian vertebrate or invertebrate animals, fungal cells, orprotozoal cells, in addition to or instead of mammalian cells. Thus,where the discussion of kits and services herein refers to mammaliancells, the invention provides embodiments that encompass non-mammalianeukaryotic cells.

VIII. Certain Aspects and Embodiments

Sortase-modified cells, e.g., sortase-modified mammalian cells, can beused to treat a wide variety of different diseases and disorders. Insome embodiments sortase-modified cells conjugated with a therapeuticagent may be used to treat any disease or condition for which theunconjugated therapeutic agent is of use. Such methods of treatment arean aspect of the invention.

In some embodiments sortase-modified cells may be administeredprophylactically, e.g., to a subject who does not exhibit signs orsymptoms of the disease or disorder for which the cells are indicated(but may be at increased risk of developing the disorder or is expectedto develop the disease or disorder). In some embodimentssortase-modified cells are administered to a subject who has developedone or more signs or symptoms of the disease or disorder, e.g., thesubject has been diagnosed as having the disease or disorder.Optionally, a method comprises diagnosing a subject as having a diseaseor disorder for which sortase-modified cells are an appropriatetreatment.

One of ordinary skill in the art will be aware of various indicationsfor therapeutic agents that may be conjugated to mammalian cells. Forexample, interferons have a variety of uses, e.g., in the treatment ofautoimmune diseases (e.g., multiple sclerosis) and certain infectiousdiseases (e.g., certain viral infections) and (often in combination withchemotherapy and radiation) as a treatment for many cancers.

In some embodiments sortagged mammalian cells or other sortaggedeukaryotic cells (e.g., sortagged eukaryotic microorganisms)administered for treatment of a disease may be used in combination withany other therapy useful in treating the disease. When entities areadministered “in combination” they may be administered in the samecomposition (if compatible) or in different compositions in variousembodiments. When administered in different compositions any order oftreatment is contemplated. Administration of or more doses of sortaggedcells may be interspersed with administration of one or more doses ofone or more other agents. Successive doses may be administered at timesseparated by time intervals of minutes, hours, days, weeks, or months.In some embodiments at least one dose of sortagged cells is administeredwithin no more than 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 22, 24, 26, 30,36, 42, 48, or 52 weeks before or after administration of one or moredoses of a different therapeutic entity, e.g., any therapeutic agentuseful in treating the disease. In some embodiments, a time intervalbetween a dose of cells and a dose of a cytotoxic or anti-proliferativedrug is selected so as to avoid significant effect, e.g., cytotoxic oranti-proliferative effect, of the cytotoxic or anti-proliferative drugon the cells.

In some embodiments sortagged mammalian cells administered for treatmentof cancer may be used in combination with a chemotherapy drug and/orradiation therapy. The cells may be administered separately from thedrug. In some embodiments cycles of drug and/or radiation therapy may beinterspersed with cycles of cell therapy. In certain embodiments it iscontemplated to administer sortagged cells in addition to any standardcancer treatment regimen, e.g., any standard chemotherapy and/orradiation regimen or instead of one or more components of such aregimen. In some embodiments a subject in need of treatment of cancermay undergo surgery to remove at least a portion of the tumor. Thesurgery may remove the entire tumor (to the extent the tumor isdetectable) or may reduce the size of the tumor but not remove theentire tumor (e.g., if the tumor is too extensive to make completesurgical removal advisable or if attempting such surgical removal isotherwise not advisable within the judgement of the skilled artisan). Insome embodiments sortagged cells may be administered to the subject oneor more times prior to surgery, one or more times during surgery, and/orone or more times after surgery. The sortagged cells may reduce the sizeof a tumor and/or may eliminate tumor cells that were not removed duringsurgery (which may be located at the site of surgery or may havedisseminated to other location(s) in the body.

Erythropoiesis stimulating agents such as EPO are of use to treatanemia, which may result from a variety of causes. For example, theanemia may be an anemia of chronic disease, anemia associated withmedications (e.g., cancer chemotherapy), radiation, renal disease (e.g.,diabetes), infectious diseases, or blood loss. Colony stimulatingfactors such as G-CSF, GM-CSF, and/or M-CSF may be used to treatleukopenia, e.g., neutropenia and/or lymphopenia, which may result,e.g., from medications (e.g., cancer chemotherapy), radiation,infectious disease, or blood loss.

Neurotrophic factor proteins may be used, e.g., to treatneurodegenerative diseases (e.g., amyotrophic lateral sclerosis,Huntington disease, Alzheimer disease, Parkinson disease), central orperipheral nervous system injury.

Interleukins are of use to modulate the immune response for a widevariety of purposes, e.g., to stimulate an immune response against aninfectious agent or cancer or to limit the intensity and/or duration ofinnate and/or adaptive immune responses. Interleukins may be of use intreatment of autoimmune diseases, sepsis, or other conditions in whichan aberrant or overactivated immune response can be deleterious.

Diseases caused by viruses, gram-positive or gram-negative bacteria,mycobacteria, fungi, or parasites are of interest in certainembodiments. For example, immune system cells may be sortagged with anagent that binds to such viruses, bacteria, fungi, or parasites, or maybe sortagged with an agent that binds to mammalian cell infected by suchviruses, bacteria, fungi, or parasites. Exemplary viruses, bacteria,fungi, and parasites are discussed above.

In general, a sortagged eukaryotic cell may be used for any purpose inwhich it is useful to have an agent attached to the surface of suchcell. In some embodiments, the agent comprises a detectable label, thusfacilitating detection of the sortagged eukaryotic cell. In someembodiments the cell is a microorganism that spends at least part of itslife cycle as an intracellular parasite of a mammalian or avian host.Examples of such microorganisms include a variety of fungi and protozoa.In some embodiments, an intracellular parasite that has been labeledwith sortase is contacted with a host cell, e.g., a mammalian or aviancell, in vitro. Processes such as attachment of the microorganism to thecell, entry of the microorganism, intracellular movement of themicroorganism, or other activities of the microorganism may be monitoredby detecting the label. In some embodiments a sortagged microorganism isof use in a method of identifying a candidate therapeutic agent fortreating a disease caused by the microorganism. For example, in someembodiments, a candidate compound is contacted with the cell, and theability of the candidate compound to inhibit one or more such activitiesis assessed. In some embodiments, if the candidate compound inhibits oneor more such activities, the candidate compound is identified as acandidate therapeutic agent for treating a disease caused by themicroorganism.

In some embodiments, a sortagged microorganism, e.g., a sortaggedeukaryotic microorganism, is used to deliver an agent to a target cell,e.g., a tumor cell, tumor-associated cell, or pathogen-infected cell. Insome embodiments the microorganism is sortagged with the agent, with atargeting moiety that binds to a molecule on the target cell, or both.In some embodiments the microorganism is one that is naturally capableof invading the target cell. For example, the microorganism may be anintracellular parasite (during at least one of its life cycle stages),e.g., an apicomplexan parasite such as T. gondii, a Trypanosomatid, aPlasmodium, a fungus such as Histoplasma capsulatum or Cryptococcusneoformans, and the target cell may be a vertebrate cell, e.g., amammalian cell, e.g., a human cell, that is susceptible to invasion bythe microorganism. Those of ordinary skill in the art will be aware ofvarious strains of microorganisms and suitable methods of obtaining andpropagating microorganisms. In some embodiments the microorganism issortagged with a targeting moiety that binds to a tumor antigen andincreases the binding of the microorganism to a cell that expresses theTA at its surface and, in some embodiments, increases subsequentinvasion of the microorganism. In embodiments in which the microorganismis sortagged with a targeting moiety, the targeting moiety may be of anyof the various types of binding moieties described herein. In someembodiments the targeting moiety comprises a single chain antibody(e.g., an scFv) or single domain antibody. For example, in someembodiments the targeting moiety comprises a VHH, some embodiments thetargeting moiety comprises a single chain antibody (e.g., an scFv) orsingle domain antibody. For example, in some embodiments the bindingmoiety comprises a VHH. In some embodiments the microorganism issortagged with an agent comprising a substance that is toxic to thetarget cell upon delivery to the surface of the cell or to the interiorof the target cell. In some embodiments the microorganism is sortaggedwith an agent comprising a substance that is toxic to the target cellupon contact with the target cell surface. In some embodiments the toxicsubstance is a chemotherapy drug, e.g., a small molecule chemotherapydrug, a toxin, a protein comprising a cytolytic domain such as granzymeor perforin, a pro-apoptotic agent such as a protein comprising apro-apoptotic domain. In some embodiments the microorganism isgenetically engineered to produce one or more molecules, e.g., asubstance that is toxic to the target cell, a cytokine, a costimulator,a targeting moiety. In some embodiments the microorganism secretes themolecule or expresses it at its cell surface. In some embodiments themicroorganism is genetically engineered to lack expression or activityof one or more endogenous gene products. One or ordinary skill in theart will be aware of appropriate methods and vectors useful for creatinggenetically engineered microorganisms. In some embodiments, a strain,e.g., a T. gondii strain, that has a deficient non-homologous endjoining pathway may be used (Fox B A, Eukaryot Cell. 2009; 8(4):520-9).For example, the strain may lack expression of a functional KU80 proteinor homolog thereof, e.g., due to a disruption or deletion in the genethat encodes KU80 or homolog thereof. In some embodiments themicroorganism is avirulent and/or can be effectively eliminated bytreating the subject with an appropriate therapeutic agent.

In some embodiments the target cell is not a tumor cell orpathogen-infected cell. The target cell may be, e.g., a normal, healthycell or an abnormal cell. In some embodiments the microorganism may besortagged with an agent that modulates one or more biological activitiesor properties of the normal or abnormal cell, e.g., in a way that isbeneficial to a subject to whom the cell is administered or in whom thecell exists. In some embodiments, the cell may be an immune system cell,and the agent may be, e.g., an immunomodulator. In some embodiments themicroorganism is genetically engineered to produce a substance, e.g., aprotein. The substance may be a therapeutic agent, enzyme, or any othersubstance the production of which is desired. In some embodiments thesubstance modulates one or more biological activities or properties ofthe cell. In some embodiments the cell is affected by a disease, and thesubstance ameliorates the effect of the disease on the cell. In someembodiments the microorganism is sortagged with a targeting moiety thatbinds to the normal or abnormal cell.

In some embodiments the microorganism is an attenuated strain.“Attenuated” refers to a strain that has reduced virulence relative to awild type strain or parental strain from which an attenuated strain isgenerated. An attenuated strain may be weakened and/or less robustcompared to a wild type strain or parental strain (i.e., a strain fromwhich the attenuated strain was derived). In some embodiments anattenuated strain is avirulent. An attenuated strain may arise naturallyand be identified by testing the virulence of the strain in a testsystem (e.g., in test cells or a test animal) or may be generated by manby, e.g., passaging the organism in a host or host cells that are not anatural host or host cell of the microorganism, by mutagenesis andselection, by irradiation, by exposure to a chemical agent, or byengineering. In some embodiments an attenuated strain has substantiallyreduced or absent ability to inflict damage on a host or host cell, hassubstantially reduced or absent ability to replicate or complete one ormore stages of its life cycle in a particular host or host cell (e.g.,humans or human cells), and/or has substantially reduced or absenttransmissibility from one host to another as compared to a wild typestrain or as compared to a parental strain. In some embodiments anattenuated strain has at least a 10-fold, 10²-fold, 10³-fold, 10⁴-fold,10⁵-fold, 10⁶-fold, 10⁷-fold, or 10⁸-fold reduced ability to replicateas compared to a wild type strain or as compared to a parental strain.In general, replication ability may be measured using any suitablemethod. In some embodiments, replication ability may be measured asnumber of new individual organisms produced or amount of DNA synthesizedby the organism and its descendants. In some embodiments an attenuatedstrain has at least a 10-fold, 10²-fold, 10³-fold, 10⁴-fold, 10⁵-fold,10⁶-fold, 10⁷-fold, or 10⁸-fold reduced ability to cause death of a hostor host cell as compared to a wild type strain or as compared to aparental strain. In some embodiments an attenuated strain ismetabolically active. In some embodiments an attenuated strain retainsability to invade host cells, e.g., mammalian host cells, e.g., humanhost cells. If desired, invasion may be quantified using an invasionassay. One of ordinary skill in the art will be aware of suitableassays. The strain's ability to invade host cells may or may not beequivalent to that of a wild type strain or parental strain. In someembodiments the invasion ability is at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of that of a wild type strain or parentalstrain. In some embodiments an attenuated strain may have a normal ornear normal level of virulence under certain conditions or in certainhosts but is avirulent or has substantially reduced virulence underother conditions, such as those existing in vivo in a subject, e.g., ahuman subject. In some embodiments, for example, an attenuated strainmay be auxotrophic for one or more nutrients. The strain may replicatenormally under conditions in which the nutrient is available (e.g.,supplied in a culture medium) but may be unable to replicate under invivo conditions in which the amount of the nutrient is insufficient tosupport replication. In some embodiments the nutrient is a precursor fornucleotide biosynthesis, e.g., a precursor for synthesis of a purine orpyrimidine. Attenuated strains are known in the art. For example,attenuated T. gondii strains are described in U.S. Pat. Pub. Nos.20100203085 and/or 20120045477, which also describe methods and vectorsuseful for genetic engineering of T. gondii. In some embodiments anattenuated T. gondii strain has a defect in de novo pyrimidinesynthesis, pyrimdine salvage, and/or transport of pyrimidine bases ornucleosides. For example, one or more genes in the de novo pyrimidinesynthesis pathway, pyrimidine salvage pathway, or a transporter ofpyrimidine bases or nucleosides may be disabled, e.g., by targetedinsertional mutagenesis. In some embodiments the gene encodes carbamoylphosphate synthetase II, aspartate transcarbamylase, dihydroorotase,dihydroorotase dehydrogenase, orotate phosphoribosyltransferase, ororotidine 5′-monophosphate decarboxylase, uridine phosphorylase (UP),uracil phosphoribosyltransferase, or purine nucleoside phosphorylase. Insome embodiments an attenuated strain is the cps strain of T. gondii,which has a knockout of the gene that encodes carbamoyl phosphatesynthetase II and exhibits uracil auxotrophy and extremely reducedvirulence (B. A. Fox, D. J. Bzik. Nature. 2002; 415:926-929). The cpsstrain invades and replicates normally in vitro if uracil is supplied inthe culture medium. As uracil is not present at an adequateconcentration to support replication of the cps strain in mammals ormammalian cells, it invades mammalian host cells normally but does notreplicate and exhibits greatly decreased virulence. In some embodimentsa subject to whom a sortagged attenuated microorganism is administeredis an individual who is infected with a non-attenuated strain of themicroorganism. A variety of microorganisms have been used or proposedfor use as live, attenuated vaccines, e.g., for protection againstinfection by non-attenuated strains of the microorganism, are known inthe art. The present disclosure contemplates sortagging of any suchmicroorganism.

In some embodiments a sortagged microorganism is contacted with targetcells ex vivo. In some embodiments a sortagged microorganism isadministered to a subject and encounters target cells in the subject. Insome embodiments a subject to whom a sortagged attenuated microorganismis administered is an individual who is infected with a non-attenuatedstrain of the microorganism. In some embodiments, a sortaggedmicroorganism is administered to a subject in need of treatment for atumor. The microorganism may be sortagged with a targeting moiety thatbinds to a tumor antigen expressed by tumor cells in the tumor. Ingeneral, the tumor cells and tumor may be of any type. In someembodiments the tumor is an ovarian cancer, colon cancer, liver cancer,prostate cancer, lung cancer, bladder cancer, breast cancer, braincancer, lymphoma, or melanoma. In some embodiments a subject to whom asortagged microorganism is administered has a tumor composed at least inpart of cells that the microorganism is capable of invading.

In some embodiments, tumor cells or pathogenic organisms, e.g.,pathogenic eukaryotic organisms, are sortagged with an agent comprisinga binding moiety that binds to a cell surface molecule expressed by anantigen presenting cell. In some embodiments the APC is a professionalAPC, such as a dendritic cell or macrophage. In general, the tumor cellsmay be derived from a tumor of any tumor type. In general, a pathogenicorganism may be any organism capable of causing disease in a mammal,e.g., a human. In some embodiments the organism is a fungal cell orparasite. In some embodiments the organism is a microorganism. In someembodiments the microorganism is T. gondii. Those of ordinary skill inthe art will be aware of various strains and suitable methods ofobtaining and propagating pathogenic organisms. In some embodiments thebinding moiety serves as a targeting moiety to target the tumor cells orpathogenic organism to APC. In some embodiments the APC is a phagocyticcell. In some embodiments the cell surface molecule is a receptorexpressed by the APC. In some embodiments the cell surface molecule isan MHC Class II molecule, CD205 (DEC205), DNGR-1 (CLEC9A), CD207(Langerin), CD11c, CD141, CD303, CD103, CD209 (DC-SIGN), CD68, or CD163.In general, the binding moiety may be of any of the various types ofbinding moieties described herein. In some embodiments the bindingmoiety comprises a single chain antibody (e.g., an scFv) or singledomain antibody. For example, in some embodiments the binding moietycomprises a VHH, e.g., a VHH that binds to human MHC Class II or a VHHthat binds to human DEC205. VHH4 is an exemplary VHH that binds to humanMHC Class II (described in WO/2013/155526). Exemplary monoclonalantibodies that bind to human DEC205 are described in Park, C J, et al.,J Immunol Methods. 2012; 377(1-2):15-22. In some embodiments the tumorcells are obtained from a subject suffering from a tumor. The tumorcells may be obtained by biopsy or surgery to remove at least a portionof the tumor or in a blood sample or other biological sample obtainedfrom the subject. In some embodiments the tumor cells are from a tumorcell line, which may be derived from tumor cells obtained from a subjectin need of treatment for a tumor or may be derived from a tumor of thesame type as that of a subject. In some embodiments the tumor cells mayexpress at least one tumor antigen.

In some embodiments tumor cells or pathogens that have been sortaggedwith a binding moiety that binds to a cell surface molecule expressed byan APC are processed. In some embodiments, processing renders the tumorcell non-viable, less viable (reduced lifespan), or incapable ofproliferating. In some embodiments the processing renders the pathogennon-viable, less viable, attenuated, non-infectious, and/or incapable ofproliferating. For example, the tumor cell or pathogen may be exposed toradiation or a toxic substance such as a pro-apoptotic agent. In someembodiments the processing may comprise physical or chemicalfragmentation, lysis, or fractionation. For example, cells may besonicated, subjected to bead beating, dounced, sheared, subjected toconditions of high or low osmolarity sufficient to induce cell lysis,exposed to a detergent, and/or exposed to a cytolytic agent. In someembodiments a cell membrane fraction may be isolated from the sortaggedtumor cells or pathogens. In some embodiments, tumor cells or pathogensmay be processed in any of the afore-mentioned ways prior to sortagging.Without wishing to be bound by any theory, fragmentation, lysis, orfractionation may produce portions of cells that are more readilyphagocytosed or endocytosed than a whole cell.

In some embodiments a tumor cell or pathogen that has been sortaggedwith a binding moiety that binds to a cell surface molecule expressed byan APC is used to deliver an antigen expressed by the tumor cell orpathogen to APC that express the cell surface molecule. By binding tothe APC, the binding moiety maintains the sortagged tumor cell orpathogen in close proximity to the APC, which may then internalize thetumor cell or pathogen or a portion thereof or a product thereof such asa molecule shed or released from the tumor cell or pathogen. Forexample, the APC may internalize the tumor cell, pathogen, or a portionor product thereof by phagocytosis or endocytosis. In some embodiments apathogen is able to invade the APC and is thereby internalized. The APCmay process the internalized tumor cell, pathogen, or portion or productthereof and present one or more antigens derived from the tumor cell,pathogen, or portion or product thereof on its surface in associationwith an MHC Class I or Class II molecule. For example, the APC maypresent a peptide comprising one or more epitopes derived from the tumorcell, pathogen, or portion of product thereof. In some embodiments tumorcells or pathogens that have been sortagged with a binding moiety thatbinds to a cell surface molecule expressed by an APC are administered toa subject, whereupon the binding moiety binds to APCs in the subject. Insome embodiments tumor cells or pathogens that have been sortagged witha binding moiety that binds to a cell surface molecule expressed by anAPC may be contacted with APCs ex vivo, e.g., by placing them in thesame vessel, whereupon the binding moiety attached to the tumor cells orpathogens binds to the APCs. In some embodiments the APCs maysubsequently be administered to a subject or may be contacted ex vivowith T cells that are subsequently administered to the subject. The APCmay present the antigen to T cells ex vivo or in a subject. Presentationof the antigen by the APC may elicit or enhance a T cell response in thesubject or ex vivo. The response may be directed toward cells thatexpress the antigen, such as tumor cells or pathogens in the subject. AnAPC may thus elicit or promote an immune response against a tumor orpathogen in a subject. In some embodiments an APC may stimulate T cellsby presenting an antigen derived from the sortagged tumor cell orpathogen to such cells. In some embodiments the T cells are naïve Tcells. In some embodiments an APC may stimulate a cytotoxic T cellresponse, a helper T cell response, or both.

In some embodiments a tumor cell or pathogen that has been sortaggedwith a binding moiety that binds to a cell surface molecule expressed byan APC is also sortagged with a second agent. In some embodiments thesecond agent is capable of stimulating the functional maturation or atleast one biological activity of an APC. In some embodiments the secondagent comprises an adjuvant. In some embodiments a tumor cell orpathogen that has been sortagged with a binding moiety that binds to acell surface molecule expressed by an APC is administered in combinationwith an adjuvant. The adjuvant may be in the same composition or may beadministered separately.

In some embodiments a subject to whom sortagged tumor cells or portionsthereof, sortagged pathogens or portions thereof, or APCs that have beencontacted ex vivo with sortagged tumor cells, pathogens, or portionsthereof, are administered is in need of treatment for a tumor or is inneed of treatment for an infection caused by the pathogen. In someembodiments, the sortagged tumor cells or tumor cell portions arederived from the subject's tumor or from a tumor of the same type asthat for which the subject needs treatment. For example, a subject inneed of treatment for a melanoma may be treated with sortagged melanomacells; a subject in need of treatment for a colon carcinoma may betreated with sortagged colon carcinoma cells, etc. In some embodimentstumor cells or pathogens that have been sortagged with a binding moietythat binds to a cell surface molecule expressed by an APC are processedprior to administration to a subject or prior to contacting them withAPCs in vitro. In some embodiments tumor cells or pathogens areprocessed, and the resulting portions are sortagged, beforeadministration to a subject or contacting with APC in vitro. Processingmay comprise physical or chemical fragmentation, lysis, orfractionation, e.g., as described above. In some embodimentsadministration of tumor cells or pathogens or portions thereof that havebeen sortagged with a binding moiety that binds to APC results inaccumulation of the tumor cells, pathogens, or portions thereof, inlymphoid tissue, e.g., in lymph nodes or other lymphoid organs. Thetumor cells, pathogens, or portions thereof may come in contact withadditional APC, or other immune system cells, in the lymphoid tissue. Insome embodiments one or more doses of tumor cells or portions thereofthat have been sortagged with a binding moiety that binds to a cellsurface molecule expressed by an APC are administered to a subject priorto, concurrently with, and/or after surgery, radiation, or chemotherapyfor treatment of a tumor.

In some embodiments, methods described above in which a eukaryoticorganism is sortagged with an agent comprising a binding moiety thatbinds to an APC are applied using a eukaryotic organisms that is notpathogenic but expresses one or more proteins that is also expressed bya pathogenic eukaryotic organism or contains one or more epitopes ofsuch a protein. In some embodiments cells derived from a multicellularpathogenic eukaryotic organism are used. It should be noted that thetumor cells or pathogenic eukaryotic organisms may in some embodimentsbe genetically engineered to produce one or more molecules, e.g., acytokine, targeting moiety, costimulator, antigen, and/or toxicsubstance.

Sortagged tumor cells, microorganisms, pathogens, or portions thereof,may be prepared under appropriate conditions (e.g., in compliance withGood Manufacturing Practices) such that the resulting preparation issuitable for administration to a mammalian subject, e.g., a humansubject. The sortagged tumor cells, microorganisms, pathogens, orportions thereof, may be mixed with a pharmaceutically acceptablecarrier. Sortagged tumor cells or portions thereof, sortagged eukaryoticpathogens or portions thereof, may be administered to a subject usingany suitable administration route. In some embodiments such cells orportions thereof are administered intravenously, orally, by inhalation,or topically. In some embodiments such cells or portions thereof areadministered locally, e.g., directly to a tumor site, e.g., by injectioninto a tumor or intraperitoneally in the case of a peritoneal tumor, orlocally to a site of infection (e.g., the lungs, liver, etc.) The numberof cells to be admininstered in a dose and the number of doses to beadministered can be determined using routine procedures known to thoseof ordinary skill in the art. In certain embodiments sortagged tumorcells or portions thereof, sortagged eukaryotic pathogens or portionsthereof, are administered to a subject in combination with sortaggedimmune system cells, e.g., sortagged T cells.

The following working examples are intended to describe exemplaryreductions to practice of certain methods, reagents, and compositionsprovided herein and do not limit the scope of the invention.

EXAMPLES Example 1: Sortase-Catalyzed Conjugation of Biotin Probe toNon-Genetically Engineered Mammalian Cells

Mouse red cell-depleted splenocytes were isolated using standardmethods. Approximately 2 million cells were incubated with abiotin-LPETG probe (1 mM) either with or without sortase A (150 μM) fromStaphylococcus aureus in 100 microliters DME without serum at 4° C. for1 hour. Cells were then washed with PBS 5 times and lysed in Laemmlibuffer. Samples were run on an SDS-PAGE gel. Approximately one quarterof the lysate from each incubation was loaded per lane. Biotinylatedproteins were visualized by blotting with streptavidin-HRP usingstandard methods. The resulting immunoblot is shown in FIG. 2. The blotdemonstrates labeling of a number of proteins with the biotin-LPETGprobe. Based on other experiments, the prominent band of about 30 kD isbelieved to be sortase itself labeled with biotin-LPETG, while the bandat about 60 kD is likely to be biotin-labeled sortase dimer.

Flow cytometry analysis was used to confirm sortase-catalyzed labelingof non-genetically engineered mammalian cells with the biotin-LPETGprobe (1 mM). Mouse red cell-depleted splenocytes isolated usingstandard methods were incubated with biotin-LPETG probe with or withoutsortase A (200 μM) from Staphylococcus aureus in DME without serum at 4°C. for 1 hour. Cells were then washed with PBS 5 times, incubated withphycoerythrin (PE)-conjugated streptavidin, and subjected to flowcytometry. Results are shown in FIG. 3. Blue histograms (indicated witharrows) show PE signal gated on living cells incubated with (right) orwithout (left) sortase A. Black histograms (no arrow) show backgroundstaining on control splenocytes. The results show specific binding ofPE-conjugated streptavicdin only to the cells that had been incubatedwith the biotin-LPETG probe in the presence of sortase, thusdemonstrating the labeling of these cells by sortase-catalyzedconjugation of the probe thereto.

The production and purification of sortase used in this experiment isdescribed in Popp, M W, et al., Nat Chem Biol. 2007; 3(11):707-8. Epub2007 Sep. 23.

The biotin-LPETG probe was prepared according to the following protocol,which also describes preparation of a TAMRA-LPETG probe. These probeshave two Gs at the C-terminus, i.e., biotin-LPETGG and TAMRA-LPETGG.

A) TAMRA-LPETGG Probe

Note: Use Fmoc-Ala-OH in place of Fmoc-Gly-OH to make probes for S.pyogenes sortase A

Resin Preparation Timing 15 Min

i Add 100 μmol of Rink amide resin (167 mg, 0.6 mmol/g) into a cappedglass column with a fritted glass filter bottom, solvate the resin indichloromethane (DCM) (7 mL) by shaking for 15 min in a wrist-actionshaker and remove the DCM by vacuum filtration.

Deprotection Timing 30 Min

ii Add 20% piperidine solution in N-methyl-2-pyrrolidone (NMP) (7 mL)and shake for 15 min to remove the resin's Fmoc protecting groups.Note: In all steps NMP may be replaced with DMF.iii Remove the piperidine solution by vacuum filtration and wash theresin three times with NMP (7 mL, 1 min), three times with DCM (7 mL, 1min) and an additional time with NMP (1 min).

Coupling Reaction Timing 2-3 h Until Pause Point, 3.5 h Per CouplingCycle

iv Dissolve Fmoc-Gly-OH (89 mg, 300 mol), HBTU (114 mg, 300 mol), andDIPEA (104 μL, 600 μmol) in NMP (7 mL) and add to the resin. Shake thesuspension for 2 h at room temperature.v Remove the reaction solution by vacuum filtration and wash the resinthree times with NMP (7 mL, 1 min) and three times with DCM (7 mL, 1min). The coupling reaction can be confirmed by performing a Kaisertest.Note: If the reaction is incomplete repeat steps iv-v with half theamount of reagents used for a standard coupling and shake for 1 h.PAUSE POINT: The resin can be stored at 4° C. after drying under vacuum.At this stage, store peptides in their Fmoc-protected form, if storageis desired.vi Repeat steps i-v with Fmoc-Gly-OH (89 mg, 300 mol), Fmoc-Thr(OtBu)-OH(119 mg, 300 mol), Fmoc-Glu(OtBu)-OH (127 mg, 300 mol), Fmoc-Pro-OH (101mg, 300 mol), Fmoc-Leu-OH (106 mg, 300 mol), Fmoc-c-aminocaproic acid(85 mg, 300 mol).Note: The Kaiser test does not work for verifying the extent of the Leucoupling, since the N-terminus of Pro is a secondary amine. To test thiscoupling reaction, one can use the chloroanil test or microcleavage.Note that the orthogonal protecting groups may not be fully removedduring this abbreviated cleavage.vii After removing the Fmoc on the ε-aminocaproic acid residue, add asolution of 5(6)-TAMRA (52 mg, 120 mol), PyBOP (63 mg, 120 mol), andDIPEA (42 μL, 240 mol) in NMP (7 mL) and shake overnight at roomtemperature. To prevent photobleaching of the fluorophore, wrap thecolumn in aluminum foil.viii Repeat step v and perform the Kaiser test to check the TAMRAcoupling.Cleavage from Resinix Suspend the resin in cleavage solution consisting of 95% TFA, 2.5%H₂O, and 2.5% TIS (5 mL) for 2 h at room temperature.x Elute the cleavage solution into 90 mL of ice cold (−0° C.) diethylether and rinse the resin with an additional 3 mL of the cleavagesolution into the ether.xi Store the ether solution at −20° C. for 20 min to precipitate thepeptide. Centrifuge the suspension at 1,900 g for 15 min at 4° C.,decant the supernatant and gently evaporate the remaining ether underreduced pressure.Due to the flammable and volatile nature of diethyl ether it isdesirable to use a spark-free freezer and centrifuge.Pause point: The crude peptide can be stored as a solid at −20° C.Critical step: The identity and purity can be verified by LC/MS analysis(linear gradient 5→45% LC/MS buffer B over 10 min). If LC/MS shows thatthe crude peptide is of sufficient purity, the next steps (xii-xiv) maybe omitted and the peptide may be used directly in sortase reactions.

HPLC Purification

xii Dissolve the dried peptide in H₂O (2 mL) and centrifuge at 14,000rpm for 10 min in a tabletop centrifuge to remove particulate matter.Note: Up to 50% of tert-butanol may be added to peptides that do notdissolve in pure H₂O. Also spin filters or syringe filters may be usedto remove particulate matter.xiii Purify the centrifuged supernatant by reverse-phase HPLC on a C18column using a 10-70% buffer B gradient over 15 min, followed byflushing at 90% buffer B for 5 min. We recommend a preliminary small 100μL injection and adjusting the gradient accordingly for peptide purityand ease of separation. Once a good gradient is established, theremaining crude material may be purified with 400-600 μL injections.xiv Analyze the fractions for product by LC/MS and lyophilize thedesired fractions to dryness.Note: TAMRA containing probes consist of a mixture of regio-isomers thatwill likely result in two product peaks during reverse phase HPLCpurification. The different isomers have no effect on labeling.The identity and purity can be verified by LC/MS analysis (lineargradient 5→45% LC/MS buffer B over 10 min) and NMR spectroscopy.Pause point: The lyophilized peptide can be stored at −20° C.indefinitely.

B) Biotin-LPETGG Probe

i Use the same reaction conditions as for synthesis of the TAMRA-LPETGGprobe through the Fmoc deprotection step of the Leu residue. At thispoint, add a solution of biotin (74 mg, 300 μmol), HBTU (114 mg, 300mol) and DIPEA (104 μL, 600 mol) in NMP (7 mL); shake for 2 h.ii Remove the reaction solution by vacuum filtration, wash the resin,and check the success of biotin coupling with a Kaiser test (remainingfree amines).iii Cleave the product from the resin as indicated in steps ix-xi forthe TAMRA probe.iv Purify by reverse phase HPLC as indicated in steps xii-xiv of theTAMRA probeThe identity and purity can be verified by LC/MS analysis (lineargradient 5→45% B in 10 min) and NMR spectroscopy.Pause point: The lyophilized peptide can be stored at −20° C.indefinitely.

Example 2: Sortase-Catalyzed Conjugation of VHH Protein toNon-Genetically Engineered Mammalian Cells

Approximately 2 million mouse red cell-depleted splenocytes isolatedusing standard methods were incubated with a GFP-specific VHH thatcontains a C-terminal LPETG (100 μM) (see Kirchhofer, A., et al., NatStruct Mol Biol. 2010 January; 17(1):133-8. doi: 10.1038/nsmb.1727. Epub2009 Dec. 13 for description of the original GFP-specific VHH, whichdoes not contain a C-terminal LPETG), either with or without sortase A(200 μM) from Staphylococcus aureus, in DME without serum at 4° C. for 1hour. Cells were then washed with PBS 5 times, incubated with GFP for 30minutes and subjected to flow cytometry. Results are shown in FIG. 4.Blue histograms show GFP signal gated on living cells that had beenincubated with (right) or without (left) sortase A. Black histogramsshow background staining on control splenocytes incubated with GFP. Theblue and black histograms in the left panel are virtuallysuperimposable. The blue histogram in the right panel is indicated withan arrow. The results show specific binding of GFP only to the cellsthat had been incubated with the GFP-specific VHH in the presence ofsortase, demonstrating the labeling of these cells by sortase-catalyzedconjugation of the VHH thereto.

Example 3: Sortase-Catalyzed Conjugation of VHH Protein toNon-Genetically Engineered Mammalian Cells

Lymphocytes are isolated from mice using standard methods, and expandedand activated in vitro using appropriate antibodies (e.g., soluble orimmobilized anti-CD3 mAb) and/or cytokines (e.g., IL-2) for T cellexpansion and activation. An aliquot of the expanded and activated cellsis incubated in culture medium with either a human tumor antigen(TA)-specific VHH (anti-TA VHH) containing a C-terminal LPETG or ananti-GFP VHH containing a C-terminal LPETG, either with or withoutsortase A from Staphylococcus aureus at 4° C. for 1 hour. Cells are thenwashed with PBS 5 times, incubated with a recombinant tumor antigenlabeled with fluorescein isothiocyanate (FITC) fluorescent dye, andsubjected to flow cytometry. Staining of the cells for FITC is comparedwith staining of control cells that had been incubated with the labeledrecombinant tumor antigen but not with the VHH. Increased FITC signalfrom the cells that had been incubated with tumor-antigen specific VHHand sortase, followed by incubation with labeled tumor antigen, ascompared with staining of control lymphocytes either (i) incubated withtumor-antigen specific VHH in the absence of sortase followed byfollowed by incubation with labeled tumor antigen; or (ii) incubatedwith anti-GFP VHH in the presence of sortase followed by incubaton withlabeled tumor antigen indicates successful sortase-mediated conjugationof the tumor-antigen specific VHH to the cells.

Cytotoxic activity of lymphocytes labeled with either anti-TA VHH oranti-GFP VHH towards target cells expressing the tumor antigen at theirsurface is assessed in vitro using standard methods such as chromiumrelease assays and compared with cytotoxic activity of controllymphocyes.

Lymphocytes labeled with either anti-TA VHH or anti-GFP VHH areadministered to separate groups of mice bearing xenografts of humantumor cells expressing the tumor antigen at their surface. Tumors areisolated after 2-6 weeks and their size and weight determined andcompared among groups.

Example 4: Sortase-Catalyzed Conjugation of VHH Protein toNon-Genetically Engineered Mammalian Cells

Lymphocytes are isolated from mice using standard methods, expanded andactivated in vitro using appropriate antibodies (e.g., soluble orimmobilized anti-CD3 mAb) and/or cytokines (e.g., IL-2) for T cellexpansion and activation, and incubated in culture medium with a humantumor antigen-specific VHH containing a C-terminal HA-LPETG or aGFP-specific VHH containing a C-terminal HA-LPETG, either with orwithout sortase A from Staphylococcus aureus at 4° C. for 1 hour. Cellsare then washed with PBS 5 times, incubated with anti-HA antibodylabeled with fluorescein isothiocyanate (FITC) fluorescent dye, andsubjected to flow cytometry. Staining of the cells for FITC is comparedwith staining of control cells that had been incubated with the labeledrecombinant tumor antigen but not with the VHH. Increased FITC signalfrom the cells that had been incubated with tumor-antigen specific VHHand sortase followed by incubation with labeled tumor antigen, ascompared with background staining of control cells incubated withtumor-antigen specific VHH in the absence of sortase followed byfollowed by incubation with labeled tumor antigen indicates successfulsortase-mediated conjugation of the tumor-antigen specific VHH to thecells.

Cytotoxic activity of lymphocytes with either HA-tagged anti-TA VHH orHA-tagged anti-GFP VHH conjugated thereto towards target cellsexpressing the tumor antigen at their surface is assessed in vitro usingstandard methods such as chromium release assays and compared.

Lymphocytes with HA-tagged anti-TA VHH or HA-tagged anti-GFP VHHconjugated thereto are administered to mice bearing xenografts of humantumor cells expressing the tumor antigen. Tumors are harvested 2 hourslater and analyzed for presence of administered lymphocytes usingimmunohistochemistry, staining with antibodies against either the HAtag, CD3 (to detect T cells), or both. Increased number of T cells inthe tumors of mice to which the lymphocytes sortagged with HA-taggedanti-TA VHH were administered as compared with the number of T cells inthe tumors of mice to which lymphocytes sortagged with HA-taggedanti-GFP VHH were administered demonstrates tumor targeting of thelymphocytes

In another experiment lymphocytes with either HA-tagged anti-TA VHH orHA-tagged anti-GFP VHH conjugated thereto are administered to separategroups of immunocompromised mice bearing xenografts of human tumor cellsexpressing the tumor antigen at their surface. Tumors are isolated after2-6 weeks and their average and total size and weight are determined andcompared among groups. Reduced average and/or total tumor size andweight in the mice to which the lymphocytes sortagged with the anti-TAVHH were administered as compared with the mice to which the lymphocytessortagged with anti-GFP VHH were administered is indicative thatsortagging with a tumor-targeting moiety can improve efficacy ofadoptive anti-tumor immunotherapy.

Example 5: Sortase-Catalyzed Conjugation of VHH Protein toNon-Genetically Engineered Human Lymphocytes

Peripheral blood mononuclear cells (PBMC) from a human donor areisolated using standard methods, expanded and activated in vitro usingappropriate antibodies (e.g., soluble or immobilized anti-CD3 mAb)and/or cytokines (e.g., IL-2) for T cell expansion and activation. Analiquot of the expanded and activated cells is incubated in culturemedium with a human tumor antigen-specific VHH containing a C-terminalLPETG or an anti-GFP VHH containing a C-terminal LPETG, either with orwithout sortase A from Staphylococcus aureus at 4° C. for 1 hour. Cellsare then washed with PBS 5 times, incubated with a recombinant tumorantigen labeled with either fluorescein isothiocyanate (FITC) or Cy7.5near-infrared fluorescent dye, and subjected to flow cytometry. Stainingof the cells for FITC or Cy7.5 is compared with staining of controlcells that had been incubated with the labeled recombinant tumor antigenbut not with the VHH. Increased FITC or Cy7.5 signal from the cells thathad been incubated with tumor-antigen specific VHH and sortase followedby incubation with labeled tumor antigen, as compared with backgroundstaining of control cells incubated with tumor-antigen specific VHH inthe absence of sortase followed by followed by incubation with labeledtumor antigen indicates successful sortase-mediated conjugation of thetumor-antigen specific VHH to the cells.

Cytotoxic activity of lymphocytes labeled with either human anti-TA VHHor anti-GFP VHH towards target cells (e.g., human tumor cells)expressing the tumor antigen at their surface is assessed in vitro usingstandard methods such as chromium release assays.

Lymphocytes labeled with either human anti-TA VHH or anti-GFP VHH areadministered to immunocompromised mice bearing human tumor xenograftsthat express the TA at their surface. Tumors are isolated after 2-6weeks and their average and total size and weight are determined andcompared among groups. Reduced average and/or total tumor size andweight in the mice to which the lymphocytes sortagged with the anti-TAVHH were administered as compared with the mice to which the lymphocytessortagged with anti-GFP VHH were administered is indicative thatsortagging with a tumor-targeting moiety can improve efficacy ofadoptive anti-tumor immunotherapy.

Example 6: Sortase-Catalyzed Conjugation of Antibodies toNon-Genetically Engineered Mammalian Lymphocytes

Examples 3-5 are repeated except that a conventional human antibodycomprising a chain that contains a sortase recognition sequence is usedinstead of a VHH.

Example 7: Sortase-Catalyzed Conjugation of Antibodies toNon-Genetically Engineered Mammalian Lymphocytes

Examples 3-5 are repeated except that an scFv comprising a sortaserecognition sequence is used instead of a VHH.

Example 8: Sortase-Catalyzed Conjugation of Antibodies toNon-Genetically Engineered Mammalian Lymphocytes

Examples 3-7 are repeated with the additional step(s) of (i) enrichingfor CD8+ cells using MACS beads prior to sortagging (using, e.g., CD8+ TCell Isolation Kit, human (#130-096-495), Miltenyi Biotec); (ii)applying co-stimulation using an antibody to CD28 prior toadministration of the sortagged lymphocytes; and/or (iii) assessingcytotoxic activity of a sample of the sortagged lymphocytes in vitro bymeasuring secretion of granzyme and/or perforin and/or by assessingCD107 cell surface expression using anti-CD107 mAbs.

Example 9: Sortase-Catalyzed Conjugation of Sortase Substrate toGenetically Unmanipulated Eukarotyic Cells of Diverse Species

Antibodies used in Examples 9, 10, 11, and/or 28: Anti-PGK (clone22C₅D8, Invitrogen), anti-mouse/human actin (clone Ab-5, BDbiosciences), anti-Toxoplasma gondii actin, horseradishperoxidase-conjugated goat anti-rabbit Ig (Southern Biotech, cat. number4041-05), horseradish peroxidase-conjugated anti-mouse Ig (GEHealthcare, cat. number NXA931), anti-TCRbeta (clone H57, BDPharmingen), anti-CD4 (clone GK1.5, ebiosciences), anti-CD19 (clone 1D4,BD Pharmingen), anti-TER119 (clone TER-119, BD Pharmingen),allophycocyanin-conjugated streptavidin (ebiosciences, cat. number17-4317), phycoerythrin-conjugated streptavidin (Southern Biotech, cat.number 7100-09S). Propidium iodide (Sigma-Aldrich, cat. number P4864).

Results:

Saccharomyces cerevisiae (W303), Toxoplasma gondii, HEK 293 T cells, ortotal mouse splenocytes from WT C57BL/6 mice were incubated 1 hour atroom temperature with or without 500 μM of biotin-LPETG and with orwithout 20 μM of a Ca2+-independent sortase A (see description inExample 11). HEK 293T cells were incubated at 20 million per milliliter,Toxoplasma gondii at 20 to 40 million per milliliter, and yeast at 6 OD280 units per milliliter. Conjugation of biotin-LPETG probes wasanalyzed by SDS page followed by Western blotting using StreptavidinHRP. Results are presented in FIGS. 5(A)-(D). The right lane of eachblot contains a number of bands clearly showing the sortase-catalyzedlabeling of multiple proteins in each cell type.

Total mouse splenocytes (20-100 million cells per milliliter) from WTC57BL/6 mice were incubated 1 hour at room temperature with 500 μM ofbiotin-LPETG and with (dark grey histograms) or without (light greyhistograms) 20 μM of sortase A. Conjugation of biotin-LPETG was analyzedby flow cytometry using fluorescently labeled streptavidin together withantibody specific for T (TCRb), B (CD19), or red cells (Ter119). Resultsare presented in FIG. 5(E) and clearly show the presence of labeledcells of each cell type. Biotin-LPETG probes labeled T and B cellsequally well, and slightly less efficiently, red cells.

We measured by flow cytometry the kinetics with which biotin-LPETG wasconjugated to red cell-depleted splenocytes by flow cytometry.Conjugation reached ˜30% of maximum after 5 minutes and ˜60% of maximumafter 15 minutes (FIG. 5(F)). Collectively, our data show that all cellstested were efficiently sortagged in a time frame compatible with theinvestigation of many biological processes. Presumably the vast majorityof cells have naturally exposed glycines at their cell surface and willtherefore be amenable to direct sortagging.

Example 10: Measurement of Immune System Cell-Mediated CytotoxicityTowards Specific Target Cells

This example demonstrates that red cell-depleted splenocytes from OTIRag^(−/−) mice contain a cell population capable of exertingcell-mediated toxicity towards appropriate target cells upon stimulationand presents a method of quantifying the cytotoxic effect. Splenocytesare a mixed population of immune system cells containing a variety ofcell populations such as lymphocytes, NK cells, and macrophages. OTIRag^(−/−) mice are deficient for Rag and are transgenic for a T cellreceptor that recognizes the SIINFEKL peptide in the context of H2^(b).These mice produce CD8⁺ cells that are specific for SIINFEKL. Due to theRag-deficient status of the mice, they lack B cells and CD4⁺ T cells.

Splenocytes were isolated from OTI Rag^(−/−) mice and depleted ofsplenocytes by osmotic shock using standard methods. Red cell-depletedsplenocytes from OTI Rag^(−/−) mice were incubated in complete RPMI(RPMI 1640 supplemented with 10% (vol/vol) inactivated FCS,β-mercaptoethanol, non essential amino acids, sodium pyruvate andpenicillin—streptomycin) in a 24 well plate coated with anti-CD3 andanti-CD28 antibody (2 μg/ml in PBS, 30 min at 37° C.) to stimulate the Tcell population. After 72 hours, cells were mixed with red-cell depletedC57BL/6 splenocytes (isogenic with the OTI Rag^(−/−) mice but for theRag mutations) that had been pre-incubated for 30 min at 37° C. incomplete RPMI plus DMSO or plus DMSO+SIINFEKL peptide (finalconcentration 1 μg/ml). The purpose of the pre-incubation in thepresence of SIINFEKL was to load cells within the C₅₇BL/6 splenocytepopulation with SIINFEKL, causing it to be displayed at the cellsurface. The cells were mixed in 96 U bottom well plates at a 1:1 ratio(200,000 each) in 200 ml complete RPMI. After 24 hours, B cell viabilitywas measured by staining the cell population with antibody to CD19 (amarker expressed on B cells) and propidium iodide (PI) and subjectingthe cells to flow cytometry. Cells that are negative for PI staining areviable.

FIG. 6 shows dot plots showing viable C57BL/6 B cells (CD19+, PI−)(indicated in the boxed regions remaining after incubation ofSIINFEKL-loaded (left panel) or control (right panel, notSIINFEKL-loaded) C57BL/6 splenocytes with the splenocytes from OTIRag−/− mice (containing CD8+ cells specific for SIINFEKL). In theabsence of SIINFEKL loading, about 7.5% of the C57BL/6 splenocytesdetected (including both viable and non-viable cells) were viable Bcells, while with SIINFEKL loading only about 1.1% of the C57BL/6splenocytes were viable B cells. SIINFKEFL loading thus resulted in adramatic reduction in the proportion of C57BL/6 splenocytes that wereviable B cells following incubation with splenocytes from OTI Rag−/−mice(1.1% with SIINFEKL loading (right panel) versus 7.5% without SIINFEKLloading (left panel). This experiment confirms that splenocytes from OTIRag−/−mice contain a population of cells that have the capacity to exertcell-mediated cytotoxicity towards target cells that bear a specifictarget antigen (in this case the peptide SIINFEKL) on their surface andthat the approach described here is suitable to measure cell-mediatedcytotoxicity towards target cells of interest.

Example 11: Non-Genetically Engineered Immune System Cells Sortaggedwith a Targeting Moiety Exhibit Cytotoxicity Specific for Target Cells

This example demonstrates that attaching a targeting moiety tonon-genetically engineered immune system cells using sortase increasestheir cytotoxicity specifically towards cells bearing a target to whichthe targeting moiety binds. As in Example 10, the experiment describedin this example makes use of red cell-depleted splenocytes from OTIRag^(−/−) mice. The splenocytes were sortagged with a VHH (VHH7) thatbinds to mouse MHC Class I, and the ability of these cells to exertcytotoxic effects towards murine B cells expressing MHC Class I wasassessed using a similar approach to that described in Example 9.

Red cell-depleted splenocytes from OTI Rag^(−/−) mice were incubated incomplete RPMI in a 24 well plate coated with anti-CD3 and anti-CD28antibody as described in Example 10. After 72 hours the cells werewashed and incubated with or without Enhancer VHH or VHH7 (500 μM, 50μM, or 5 μM) with or without sortase A (final concentration 20 μMmutated S. aureus srtA, Ca 2+ independent), in a total volume of 200 μlof PBS, 5×10⁶ cell per reaction at room temperature for 1 hour, in orderto conjugate the relevant VHH to the cell surface. The Ca2+ independentsortase that was used in this experiment is a 6×His tagged version withthe following sequence:

(SEQ ID NO: 7) MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQSLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLE HHHHHH

Enhancer VHH refers to a VHH that binds to GFP and has been modified tocontain an LPETG sequence at its C-terminus, thus permitting the VHH toserve as a sortase substrate (see also Example 2). As noted above, VHH7is a VHH that binds to murine MHC Class II molecules (PCT/US2013/036630(WO/2013/155526; Witte, M D et al. (2012) PNAS, 109(30): 11993-11998). Aversion of VHH7 that contains a C-terminal LPETG was used in thisexperiment, thus permitting the VHH to serve as a sortase substrate.Where the terms “Enhancer VHH” and “VHH7” are used below and in FIG. 7(which presents data from this Example), it should be assumed that theversions containing a C-terminal LPETG sequence were used.

Following incubation with VHH7 or Enhancer VHH (or following controlincubation without VHH), in each case either with or without sortase,red cell-depleted splenocytes from OTI Rag^(−/−) mice were incubatedwith various concentrations of purified GFP protein or without GFP(Control). Binding of GFP through conjugated Enhancer-LPETG was analyzedby flow cytometry. As shown in FIG. 7(A), GFP binds to cells that wereexposed to Enhancer in the presence of sortase (lower left panel) butdoes not bind to cells that were exposed to Enhancer in the absence ofsortase or to cells that were exposed to VHH7 in the absence or presenceof sortase (other 3 panels). To further demonstrate thesortase-catalyzed conjugation of VHHs to the cell surface, the amount ofGFP bound to cells that had been incubated under each condition (i.e.,in the absence or presence of sortase, in each case with either Enhanceror VHH7) was estimated by analyzing cell lysates by SDS-PAGE and Westernblotting against GFP protein and comparing signal to a GFP standard(right lanes of FIG. 7(B)). As shown in FIG. 7(B), GFP binds only to thecells that were incubated with Enhancer in the presence of sortase. Thenumber of VHHs installed was approximately proportional to theconcentration of VHHs used for the reaction.

We estimated the number of VHHs installed per cell by measuring thenumber of bound GFP molecules by SDSPAGE and immunoblotting, using asolution of GFP of known concentration as standard (FIG. 7(B)).Sortagging of T cells in the presence of 500 μM of VHHs and sortase Aresulted in the conjugation of ˜1 million VHHs per cell.

Following incubation with VHH7 or Enhancer VHH (or following controlincubation without VHH), in each case either with or without sortase,red cell-depleted splenocytes from OTI Rag^(−/−) mice were washed andthen incubated with red cell-depleted wild type C57BL/6 splenocytes for24 h in U bottom 96 well plates at a 1:1 ratio (200,000 each) in 200 μlcomplete RPMI. After 24 hours, B cell viability among the redcell-depleted wild type C57BL/6 splenocytes was measured by stainingwith CD19 antibody and propidium iodide (PI) and subjecting the cells toflow cytometry. As a control, viability of CD4⁺ T cells among the redcell-depleted wild type splenocytes was measured by staining with CD4antibody and propidium iodide (PI).

FIGS. 7(C) and 7(D) present bar graphs showing the percentage of viableC57BL/6 T cells (CD4+) and B cells (CD19⁺, PI⁻) in a representativeexperiment conducted with VHH7 either in the absence of sortase (leftpanel) or in the presence of sortase (right panel). The data indicatethat non-sortagged splenocytes from OTI Rag^(−/−) mice are not cytotoxictowards either C57BL/6 B cells or T cells i.e., approximately 100% ofthe C57BL/6 B cells and approximately 100% of the C57BL/6 T cells remainviable following incubation with non-sortagged splenocytes from OTIRag^(−/−) mice (regardless of whether such non-sortagged splenocytesfrom OTI Rag^(−/−) mice had been incubated with VHH7, enhancer VHH, orno VHH). The data presented in FIG. 7(C) indicate that splenocytes fromOTI Rag^(−/−) mice that have been incubated with sortase alone (control)are not cytotoxic towards either C57BL/6 B cells or T cells i.e.,approximately 100% of the C57BL/6 B cells and approximately 100% of theC57BL/6 T cells remain viable following incubation with splenocytes fromOTI Rag^(−/−) mice that had been incubated with sortase. The datapresented in FIG. 7(D) indicate that splenocytes from OTI Rag^(−/−) micethat have been incubated with sortase and Enhancer VHH are not cytotoxictowards either C57BL/6 B cells or T cells i.e., approximately 100% ofthe C57BL/6 B cells and approximately 100% of the C57BL/6 T cells remainviable following incubation with splenocytes from OTI Rag^(−/−) micethat had been incubated with sortase and Enhancer VHH. The datapresented in FIG. 7(D) also indicate that splenocytes from OTI Rag^(−/−)mice that have been incubated with sortase and VHH7 (500 micromolar or50 micromolar) are cytotoxic towards C57BL/6 B cells, but not towardsC57BL/6 T cells. Thus, approximately 100% of the C57BL/6 T cells remainviable following incubation with splenocytes from OTI Rag^(−/−) micethat had been incubated with sortase and Enhancer VHH, whereas onlyabout 40% or 60% of the C57BL/6 B cells remain viable followingincubation with splenocytes from OTI Rag^(−/−) mice that had beenincubated with sortase and 500 micromolar or 50 micromolar VHH7,respectively. These results demonstrate that non-genetically engineeredcytotoxic immune system cells can be conjugated to a targeting moiety(in this case VHH7) using sortase and, as a result of such conjugation,exert cytotoxic effects specifically towards target cells that expressthe specific target of the binding moiety (in this case MHC Class II) attheir cell surface.

Example 11A: Installation of Two Different Agents on Cells Using Sortase

To investigate whether two different probes could be installed onlymphocytes, we incubated erythrocte-depleted splenocytes with orwithout enhancer-LPETG for 60 minutes in the presence of sortase A,followed by the addition of biotin-LPETG to the reaction for 15 minutes(FIG. 11). Cells incubated with enhancer-LPETG prior to biotin-LPETG hadsimilar amounts of surface-conjugated biotin compared to cells incubatedwith biotin-LPTEG alone (FIG. 11). These data suggest that sortagging ofVHHs to cells only minimally affects subsequent conjugation ofbiotin-LPETG. It is possible that the smaller LPETG-tagged probes mayhave more ready access to surface-displayed nucleophiles left unoccupiedby larger LPETG-tagged proteins.

Example 12: Sortase Conjugation of CAR-Modified T Cells with Anti-PD-L1Agent

Human T cells obtained from a patient with B-cell chronic lymphoblasticleukemia are genetically modified to express a chimeric antigen receptor(anti-CD20 single chain Fv region fused to the transmembrane andintracellular domain of CD3-zeta, containing signaling domains ofCD3-zeta and CD28). The cells are expanded for 10 days in culture withanti-CD3/anti-CD28 beads, then incubated with sortase and an agentcomprising (i) the extracellular domain of PD-1 and (ii) a sortaserecognition sequence, and transferred back into the patient. The effectof the administered cells on the leukemia is monitored.

Example 13: Sortase Conjugation of CAR-Modified T Cells with Anti-PD-1Agent

Human T cells obtained from a patient with B-cell chronic lymphoblasticleukemia are genetically modified to express a chimeric antigen cellreceptor (anti-CD20 single chain Fv region fused to the transmembraneand intracellular domain of CD3-zeta, containing signaling domains ofCD3-zeta and CD28). The cells are expanded for 10 days in culture withanti-CD3/anti-CD28 beads, then incubated with sortase and an agentcomprising an scFv that binds to PD-1 that has been modified to comprisea sortase recognition sequence, and are then transferred into thepatient. The effect of the administered cells on the leukemia ismonitored.

Example 14: Treatment of Multiple Myeloma with Anti-CS1 or Anti-BCMAConjugated NK Cells

NK cells are isolated from a myeloma patient and expanded in culture.The cells are incubated with sortase and an anti-CS-1 or anti-BCMAantibody that has been modified to comprise a sortase recognitionsequence, and are then transferred into the patient. The treatment isrepeated weekly for 12 weeks. The effect of the administered cells onthe multiple myeloma is monitored.

Example 15: Prevention of Metastasis with TRAIL-ES Conjugated PMBCs

PBMCs from a patient recently diagnosed with stage III colon carcinomaare isolated. The cells are incubated with sortase and an anti-CS-1 oranti-BCMA antibody that has been modified to comprise a sortaserecognition sequence, and are then transferred into the patient. Thetreatment is repeated weekly for 12 weeks following colonic resection.The patient is monitored for presence of local recurrence or metastasis.

Example 16: Inhibition of Mesothelioma Tumor Growth in a Mouse Model byMesothelin-Targeted T Cells

A modified version of SS1 Fv, an Fv specific for mesothelin, isgenerated. The modified Fv has a sortase recognition sequence at theC-terminus. Human T cells (not genetically modified) are incubated withsortase and the modified SS1 Fv.

To assess the therapeutic effect of the SS1Fv-conjugated cells againstestablished mesothelioma, mesothelioma tumors are established byintraperitoneal (i.p.) injection of 5 million LMB-H226-GL cells in 200μl of growth media into the low abdomen or flank area of eight week oldfemale athymic nude mice (ATHYMIC NCr-nu/nu) as described (Feng M, etal., J Cancer 2: 123-131). LMB-H226-GL cells are a human mesotheliomacell line LMB-H226-GL generated by Feng, et al by fluorescently labelingthe NCI-H226 human mesothelioma cell line by a lentiviral vectorharboring a luciferase-GFP (Luc/GFP) fusion gene driven by the RNApolymerase II promoter. After single-cell cloning by flow cytometry, aclone (named LMB-H226-GL) that stably expresses high levels of Luc/GFPwas obtained. The labeled cells can be imaged in vivo, e.g., to monitortumor growth. Following introduction of the mesothelioma cells, animalsare imaged the following day and then once every week thereafter. Theanimals are divided into 5 groups; (1) Vehicle, (2) SS1P (0.4 mg/kg),(3) mesothelin-targeted T cells, (4) IL12-SS1 (Fv) (1.6 mg/kg bodyweight); (5) IL12-SS1 (Fv) (1.6 mg/kg body weight)+ cells. IL12-SS1 (Fv)is a recombinant immunocytokine in which IL12 is fused to theanti-mesothelin antibody scFv (SS1). The p40 and p35 subunits of murineIL12 are connected by flexible linker (Ser4Gly)3. IL12-SS1 is describedfurther in Kim, H., et al., PLoS One. 2013 Nov. 15; 8(11):e81919. doi:10.1371/journal.pone.00819190.

The tumor-bearing mice are treated with the respective treatments orvehicle every the other day. The day when the mice were injected withthe tumor cells is considered as day 1. SS1P, anti-MSLN scFv conjugatedwith Pseudomonas toxin, is used as a positive control. The treatmentgroups and control group each contain 5 mice. Each mouse in thetreatment groups receives 0.4 mg/kg body weight of SS1P, 0.4 mg/kg (lowdose group), or 1.6 mg/kg (high dose group) of IL12-SS1 (Fv) every otherday. The control group receives PBS as a vehicle control. Body weightand tumor growth are assessed twice a week.

Assessment of tumor growth is performed as follows: Two hundredmicroliters of 15 mg/mL D-luciferin (Caliper Life Sciences, Hanover,Md.) in PBS are injected i.p. before imaging. The luciferase activity ofthe tumor os calculated using Living Image 3.1.0 software (Caliper LifeSciences, Hanover, Md.). Intraperitoneal Tumor growth was assessed usingphoton intensity, photons per second (ph/sec) as luciferase activity asdescribed (Feng, et al, cited earlier in this example). At the end ofthe treatment, three mice in each group are euthanized. Blood is takenfor whole blood complete blood counts (CBC) and serum chemistryanalysis. A full necropsy is performed, in which organs and tissues areweighed and examined for gross findings. Statistical analysis isperformed with Prism (version 5) for Windows (GraphPad Software, LaJolla, Calif.). Raw data are analyzed by “analysis of variance” withDunnett's and Newman-Keuls multiple comparison post tests. p values<0.05are considered statistically significant.

Example 17: Treatment of Ovarian Cancer in a Mouse Model with Anti-CA125Antibody Conjugated NK Cells

Human NK cells are incubated with sortase and an anti-CA-125 antibodythat has been modified to comprise a sortase recognition sequence.Following conjugation, NK cells are cultured with OVCAR cells (anepithelial ovarian cancer cell (EOC) line that expresses CA125 andmesothelin). Cells are stained with propidium iodide and analyzed byFACS. The ability of the CA-125-targeted NK cells to kill OVCAR cells invitro is confirmed.

Efficacy of CA-125-targeted NK cell therapy in vivo either as singleagent or in combination with docetaxel (DTX) (a standardchemotherapeutic agent used to treat ovarian cancer) is assessed in anintraperitoneal (i.p.) EOC mouse model that utilizes a subline of theOVCAR cell line termed OVCAR-3. (Further details of the OVCAR3 cells andmodel are described in Pourgholami M H, et al. Clin Cancer Res 12:1928-1935 and Wang L, et al. PLoS ONE 6(9): e24405.doi:10.1371/journal.pone.0024405.). OVCAR-3 cells are implantedintraperitoneally in female athymic nude mice and allowed to grow tumorand ascites. Mice are then treated with various number ofCA-125-targeted NK cells, various concentrations of DTX, combinationtest (CA-125-targteted NK cells and DTX), combination control(unconjugated human NK cells and DTX) or vehicle control i.p for 3weeks. Treated mice are killed 4 weeks post-treatment. Ascites volume,tumor weight, CA125 levels from ascites, and survival of animals areassessed. The expression of MUC1 (tumor antigen expressed by OVCARcells), CD31, Ki-67 (proliferation marker), TUNEL and apoptotic proteinsin tumor xenografts was evaluated by immunohistochemistry. The abilityof CA-125-targeted NK cells to inhibit i.p. tumor development, growth,and ascites production in a dose-dependent manner is assessed.

Example 18: Treatment of Ovarian Cancer in a Mouse Model with Anti-CA125Antibody Conjugated T Cells

The preceding example is repeated using human T cells obtained from thepatient and expanded in culture.

Example 19: Treatment of Ovarian Cancer with Anti-CA125 andAnti-Mesothelin Antibody Conjugated NK Cells

NK cells are isolated from a patient with stage III primary ovariancancer and expanded in culture. An aliquot of the cells are incubatedwith sortase and an anti-CA-125 antibody that has been modified tocomprise a sortase recognition sequence. A second aliquot of the cellsis incubated with sortase and an anti-mesothelin antibody that has beenmodified to comprise a sortase recognition sequence.

Cells from each sortagged population are transferred back into thepatient. The treatment is repeated weekly for 12 weeks followingsurgery. The effect of the administered cells on residual cancer andmetastases is monitored. The patient is monitored for blood levels ofCA-125 and is retreated with CA-125-targeted NK cells if an elevatedCA-125 level is detected.

Example 20: Treatment of Ovarian Cancer with Anti-CA125 andAnti-Mesothelin Antibody Conjugated PBMCs

The preceding example is repeated except that PBMCs obtained from thepatient are used.

Example 21: Treatment of Ovarian Cancer with Anti-CA125 andAnti-Mesothelin Antibody Conjugated Allogeneic NK Cells

The preceding example is repeated except that allogeneic NK cells, inthis case NK-92 cells, are used

Example 22: Treatment of Ovarian Cancer with IL-12-Conjugated CAR TCells

Human T cells obtained from a patient with stage III ovarian cancer aregenetically modified to express a chimeric T cell receptor (anti-CA125single chain Fv region fused to the transmembrane and intracellulardomain of TCR, containing signaling domains of CD3-epsilon and CD28).The cells are expanded for 10 days in culture with CD3-CD28 beads andthen incubated with sortase and a fusion protein that contains the twosubunits of human interleukin-12 as a single polypeptide with a sortaserecognition sequence at the C-terminus, and transferred back into thepatient after surgery. Treatment is repeated weekly for 12 weeks. Theeffect of the administered cells on residual cancer and metastases ismonitored. The patient is monitored for blood levels of CA-125 and isretreated if an elevated CA-125 level is detected.

Example 23: Treatment of Ovarian Cancer with T Cells Sortagged with aBispecific Agent

Single domain antibodies specific for CA-125 and CD3 epsilon areproduced in a recombinant cell expression system. The sdAb specific forCD3 epsilon is produced with a sortase recognition motif incorporated atthe C-terminus. Click chemistry handles are installed at the N-terminiof each sdAb using sortase. The two sdAbs are then conjugated togetherto produce a bispecific antibody. Human T cells obtained from a patientwith stage III ovarian cancer are expanded in culture with CD3-CD28beads and then incubated with sortase and the bispecific antibody andare transferred back into the patient after surgery. Treatment isrepeated weekly for 12 weeks. The effect of the administered cells onresidual cancer and metastases is monitored. The patient is monitoredfor blood levels of CA-125 and is retreated if an elevated CA-125 levelis detected.

Example 24: Treatment of Leukemia with Sortagged T Cells

Human T cells obtained from a patient with B-cell acute lymphoblasticleukemia are expanded for 10 days in culture with anti-CD3/anti-CD28beads, then sortagged with an scFv that binds to CD19 and transferredinto the patient. The effect of the administered cells on the leukemiais monitored.

Example 25: Treatment of Leukemia with Sortagged T Cells

Human T cells obtained from a patient with B-cell acute lymphoblasticleukemia are genetically modified to express a chimeric antigen cellreceptor (anti-CD19 single chain Fv region fused to the transmembraneand intracellular domain of TCR, containing signaling domains ofCD3-zeta and CD28). The cells are expanded for 10 days in culture withanti-CD3/anti-CD28 beads, then sortagged with an scFv that binds toCD22, and transferred into the patient. The effect of the administeredcells on the leukemia is monitored.

Example 26: Treatment of Leukemia with Sortagged T Cells

Human T cells obtained from a patient with B-cell acute lymphoblasticleukemia are genetically modified to express a chimeric antigen cellreceptor (anti-CD22 single chain Fv region fused to the transmembraneand intracellular domain of TCR, containing signaling domains ofCD3-zeta and CD28). The cells are expanded for 10 days in culture withanti-CD3/anti-CD28 beads, then sortagged with an scFv that binds toCD19, and transferred into the patient. The effect of the administeredcells on the leukemia is monitored.

Example 27: Clinical Trial of Treatment of Ovarian Cancer withAnti-CA125 Antibody Conjugated T Cells

Patients with advanced ovarian cancer are randomized to receive,following surgery, either (1) standard adjuvant chemotherapy withplatinum-taxane alone; (2) standard adjuvant chemotherapy withplatinum-taxane and, in addition, therapy with intravenouslyadministered autologous T cells conjugated with an anti-CA-125 antibodyusing sortase without genetic modification; or (3) standard adjuvantchemotherapy with platinum-taxane and, in addition, therapy with bothintravenously and intraperitoneally administered autologous T cellsconjugated with an anti-CA-125 antibody using sortase without geneticmodification. Progression-free survival and 5 year survival rates ofpatients in the three treatment groups are monitored and compared.

Example 28: Sortase Conjugation of Red Blood Cells with Anti-PD-L1 Agent

Human red blood cells are obtained from a patient with B-cell chroniclymphoblastic leukemia or from an immunocompatible donor and incubatedwith sortase and an agent comprising (i) the extracellular domain ofPD-1 and (ii) a sortase recognition sequence. Red blood cells aretransferred into the patient after sortagging. The effect of theadministered cells on the leukemia is monitored.

Example 29: Sortase Conjugation of Red Blood Cells with Anti-PD-1 Agent

Human red blood cells obtained from a patient with B-cell chroniclymphoblastic leukemia or from an immunocompatible donor are incubatedwith sortase and an agent comprising an scFv that binds to PD-1 and thathas been modified to comprise a sortase recognition sequence. Red bloodcells are transferred into the patient after sortagging. The effect ofthe administered cells on the leukemia is monitored.

Example 30: Treatment of Multiple Myeloma with Anti-CS1 or Anti-BCMAConjugated Red Blood Cells

Red blood cells are isolated from a myeloma patient or from animmunocompatible donor. The cells are incubated with sortase and ananti-CS-1 or anti-BCMA antibody that has been modified to comprise asortase recognition sequence, and are then transferred into the patient.The treatment is repeated weekly for 12 weeks. The effect of theadministered cells on the multiple myeloma is monitored.

Example 31: Prevention of Metastasis with TRAIL-ES Conjugated RBCs

Red blood cells are isolated from a patient recently diagnosed withstage III colon carcinoma or obtained from an immunocompatible donor.The cells are incubated with sortase and an anti-CS-1 or anti-BCMAantibody that has been modified to comprise a sortase recognitionsequence, and are then transferred into the patient. The treatment isrepeated weekly for 12 weeks following colonic resection. The patient ismonitored for presence of local recurrence or metastasis.

Example 32: Treatment of EAE Using Red Blood Cells Sortagged withPeptide Fragment of Myelin Basic Protein

Sortase is used to conjugate either myelin basic protein (MBP) that hasbeen modified to comprise a sortase recognition sequence or ovalbuminthat has been modified to comprise a sortase recognition sequence tonon-genetically modified RBCs obtained from SJL mice.

Eight SJL mice are injected intravenously (iv) via tail vein with 1×10⁸RBC coupled to mouse MBP (MBP-RBC). Control mice receive 1×10⁸ovalbumin-coupled RBCs (OV-RBC), also prepared using sortase. One weeklater, all animals are immunized with syngeneic spinal cord homogenatein an emulsion in complete Freund's adjuvant to induce EAE according tostandard methods.

Animals are weighed and examined daily from Day 7. Neurological deficitis graded according to the following scale: mild, a flaccid tail for 2or more days with associated weight loss; or severe, definite paralysis,often with scissoring of the hind limbs. Animals are sacrificed 27 daysafter initial immunization. Brains are removed and fixed in 10%Formalin. Sections are made, stained with hematoxylin and eosin andscored on a scale from 1-5 in a blinded manner, according to the extentof meningeal inflammation and lymphocyte cuffing.

The clinical severity of EAE, pathologic severity of EAE, and weightloss are compared between the MBP-RBC and control groups. A lower levelof clinical severity, lower level of pathological severity, and/orreduced weight loss (or weight gain instead of weight loss) in theMBC-RBC treated group as compared with the controls is evidence ofeffective inhibition of EAE.

Example 33: Treatment of Melanoma Using Sortagged T Cells

T cells isolated from a patient diagnosed with melanoma or obtained froman immunocompatible donor are expanded and sortagged with an antibody toa melanoma antigen, for example MART-1, and two checkpoint inhibitors,for example ipilimumab (anti-CTLA4) and anti-PD1 nivolumab. Thesortagged T cells are administered to a patient with melanoma. Theeffect of the administered cells on the melanoma is monitored.

Example 34: Treatment of HER2 Positive Breast Cancer Using Sortagged RedBlood Cells

RBCs isolated from a patient diagnosed with HER2 positive breast canceror obtained from an immunocompatible donor are sortagged with Herceptin,anti-PD1 antibody, and anti-VEGFR2 antibody and are administered to thepatient with HER2 positive breast cancer. The effect of the administeredcells on the cancer is monitored.

Example 35: Treatment of HER2 Positive Breast Cancer Using Sortagged RedBlood Cells

RBCs isolated from a patient diagnosed with HER2 positive breast canceror obtained from an immunocompatible donor are sortagged with TRAIL andHerceptin and administered as an adjunct to standardchemotherapy/Herceptin therapy of HER-2 positive breast cancer. Theeffect of the administered cells on the cancer is monitored.

Example 36: Treatment of Relapsed/Refractory B-Precursor ALL

Relapsed/refractory B-precursor ALL in adult patients is an aggressivemalignant disease with dismal prognosis and unmet medical need. Redblood cells sortagged to carry blinatumomab (a bispecific single-chainantibody construct designed to link B cells and T cells resulting in Tcell activation and a cytotoxic T cell response against CD19 expressingcells) are administered to adult patients with relapsed/refractoryB-precursor ALL. Patients receive up to five monthly cycles ofintravenous RBC-blinatumomab treatment.

Example 37: Sortase-Catalyzed Installation of VHHs on Toxoplasma gondiiAllows Cell-Specific Targeting

As described above, modification of CD8 T cells through sortagging doesnot obviously interfere with cytotoxic functions. Invasion of host cellsby parasites represents another type of cell-cell interaction. Toinvestigate whether Toxoplasma gondii tachyzoites modified using sortasewould be able to invade cells, we sortagged parasites withTAMRA-modified LPETG peptides and incubated them together with humanforeskin fibroblasts. Tachyzoites (20 to 40 million per milliliter) wereincubated with 500 μM TAMRA-LPETG and 20 μM Ca2+-independent sortase A(same as used in Example 11) in HHE (Hanks buffer+Hepes+EDTA) for 15minutes. Parasites were then washed and incubated with human foreskinfibroblasts (HFF). Sortagged parasites were visualized by fluorescencemicroscopy and their ability to invade the fibroblasts was monitored andrecorded. We found that the sortagged parasites were perfectly capableof invading fibroblasts (FIG. 8(A); video available upon request).

To address whether Toxoplasma gondii could be targeted to specificcells, we sortagged parasites with biotin or biotin plus Enhancer orVHH7 and incubated them with wild type splenocytes. Toxoplasma gondiitachyzoites were incubated with or without 50 μM enhancer-LPETG orVHH7-LPETG and 20 μM Ca2+-independent sortase A (same as used in Example11) in HHE. After 20 minutes, biotin-LPETG was added for 15 minutes.Parasites were then washed and incubated with red-cell depletedsplenocytes for 1 hour at a multiplicity of infection of 5. Cells werethen washed and stained with a CD19-specific antibody and fluorescentlylabeled streptavidin. The percentages of CD19+ and CD19⁻ cells that werebiotin positive (indicative of invasion by sortagged T. gondii) werequantified by FACS. The histogram in FIG. 8(B) shows the percentage ofcells that were positive for sortagged T. gondii within the CD19negative or positive populations. Sortagging of VHH7 to Toxoplasmagondii resulted in a dramatic increase of B cells targeted by Toxoplasmagondii, together with a significant decrease of binding of non-B cellssuggesting very selective targeting. In addition, it enhanced thepercentages of B cell lysed upon infection (FIG. 8(C)). The B cellslysis assay was performed as follows: Toxoplasma gondii tachyzoites wereincubated with or without 50 μM enhancer- or VHH7-LPETG and 20 μMsortase A at room temperature in HHE buffer for 15 minutes. Afterwashing T. gondii was incubated together with 0.5 million magneticbeads-purified splenic B cells from WT or class II MHC k.o. mice at amultiplicity of infection of 5 in 100 μl or complete RPMI in 96 flatbottom well plates. After 15 hours supernatants were harvested and celllysis was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assaykit (Promega, cat. G1781) according to manufacturer's instructions.

These results support the feasibility of using sortagging to targetpathogens (e.g., cytolytic pathogens) to cells of interest, e.g., cancercells.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.The advantages and objects of the invention are not necessarilyencompassed by each embodiment of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments describedherein, which fall within the scope of the claims. The scope of thepresent invention is not to be limited by or to embodiments or examplesdescribed above.

Section headings used herein are not to be construed as limiting in anyway. It is expressly contemplated that subject matter presented underany section heading may be applicable to any aspect or embodimentdescribed herein.

Embodiments or aspects herein may be directed to any agent, composition,article, kit, and/or method described herein. It is contemplated thatany one or more embodiments or aspects can be freely combined with anyone or more other embodiments or aspects whenever appropriate. Forexample, any combination of two or more agents, compositions, articles,kits, and/or methods that are not mutually inconsistent, is provided.

Articles such as “a”, “an”, “the” and the like, may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext.

The phrase “and/or” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined. Multiple elements listed with “and/or” should be construed inthe same fashion, i.e., “one or more” of the elements so conjoined.Other elements may optionally be present other than the elementsspecifically identified by the “and/or” clause. As used herein in thespecification and in the claims, “or” should be understood to have thesame meaning as “and/or” as defined above. For example, when used in alist of elements, “or” or “and/or” shall be interpreted as beinginclusive, i.e., the inclusion of at least one, but optionally more thanone, of list of elements, and, optionally, additional unlisted elements.Only terms clearly indicative to the contrary, such as “only one of” or“exactly one of” will refer to the inclusion of exactly one element of anumber or list of elements. Thus claims that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present, employed in, or otherwiserelevant to a given product or process unless indicated to the contrary.Embodiments are provided in which exactly one member of the group ispresent, employed in, or otherwise relevant to a given product orprocess. Embodiments are provided in which more than one, or all of thegroup members are present, employed in, or otherwise relevant to a givenproduct or process. Any one or more claims may be amended to explicitlyexclude any embodiment, aspect, feature, element, or characteristic, orany combination thereof. Any one or more claims may be amended toexclude any agent, sortase substrate, sortase, composition, amount,dose, administration route, cell type, species, target, cellular marker,antigen, epitope, targeting moiety, or combination thereof. In certainembodiments cells are not CHO cells. In certain embodiments cells arenot HEK293T cells. In certain embodiments the sortase substrate used ina sortase reaction does not comprise an enhanced green fluorescentprotein. In certain embodiments the sortase substrate used in a sortasereaction does not comprise an enhanced cyan fluorescent protein (ECFP).In certain embodiments the sortase substrate used in a sortase reactiondoes not comprise an AlexaFluor. In certain embodiments cells are notpre-incubated with sortase before being contacted with a sortasesubstrate in the presence of sortase. In certain embodiments cells arenot pre-incubated with a nucleophilic acceptor sequence, e.g., anoligoglycine, e.g., triglycine, before being contacted with a sortasesubstrate in the presence of sortase. In certain embodiments if cellsare pre-incubated with sortase and/or with a nucleophilic acceptorsequence, e.g., an oligoglycine, e.g., triglycine, before beingcontacted with a sortase substrate in the presence of sortase, suchpre-incubation is for less than 15 minutes, less than 30 minutes, lessthan 60 minutes, or less than 120 minutes.

Embodiments in which any one or more limitations, elements, clauses,descriptive terms, etc., of any claim (or relevant description fromelsewhere in the specification) is introduced into another claim areprovided. For example, a claim that is dependent on another claim may bemodified to include one or more elements or limitations found in anyother claim that is dependent on the same base claim. It is expresslycontemplated that any amendment to a genus or generic claim may beapplied to any species of the genus or any species claim thatincorporates or depends on the generic claim.

Where a claim recites a composition, methods of using the composition asdisclosed herein are provided, and methods of making the compositionaccording to any of the methods of making disclosed herein are provided.Where a claim recites a method, a composition for performing the methodis provided. Where elements are presented as lists or groups, eachsubgroup is also disclosed. It should also be understood that, ingeneral, where embodiments or aspects is/are referred to herein ascomprising particular element(s), feature(s), agent(s), substance(s),step(s), etc., (or combinations thereof), certain embodiments or aspectsmay consist of, or consist essentially of, such element(s), feature(s),agent(s), substance(s), step(s), etc. (or combinations thereof). Itshould also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited. Any method of treatment may comprise a step of providing asubject in need of such treatment. Any method of treatment may comprisea step of providing a subject having a disease for which such treatmentis warranted. Any method of treatment may comprise a step of diagnosinga subject as being in need of such treatment. Any method of treatmentmay comprise a step of diagnosing a subject as having a disease forwhich such treatment is warranted.

Where ranges are given herein, embodiments in which the endpoints areincluded, embodiments in which both endpoints are excluded, andembodiments in which one endpoint is included and the other is excluded,are provided. It should be assumed that both endpoints are includedunless indicated otherwise. Unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in various embodiments, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise. “About” in reference to a numericalvalue generally refers to a range of values that fall within ±10%, insome embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%of the value unless otherwise stated or otherwise evident from thecontext. In any embodiment in which a numerical value is prefaced by“about”, an embodiment in which the exact value is recited is provided.Where an embodiment in which a numerical value is not prefaced by“about” is provided, an embodiment in which the value is prefaced by“about” is also provided. Where a range is preceded by “about”,embodiments are provided in which “about” applies to the lower limit andto the upper limit of the range or to either the lower or the upperlimit, unless the context clearly dictates otherwise. Where a phrasesuch as “at least”, “up to”, “no more than”, or similar phrases,precedes a series of numbers, it is to be understood that the phraseapplies to each number in the list in various embodiments (it beingunderstood that, depending on the context, 100% of a value, e.g., avalue expressed as a percentage, may be an upper limit), unless thecontext clearly dictates otherwise. For example, “at least 1, 2, or 3”should be understood to mean “at least 1, at least 2, or at least 3” invarious embodiments. It will also be understood that any and allreasonable lower limits and upper limits are expressly contemplated.

1-141. (canceled)
 142. A human cell having an agent linked thereto via asortase recognition sequence, wherein the agent is linked to anendogenous, non-genetically engineered protein of the human cell, andwherein the human cell has not been genetically engineered to express aprotein comprising a sortase recognition sequence, and wherein the agentcomprises a cytokine, an antigen, a costimulatory molecule, or anadjuvant.
 143. The human cell of claim 142, wherein the human cell is acellular artificial antigen presenting cell (aAPC).
 144. The human cellof claim 142, wherein the agent comprises an antigen, and wherein theantigen is a tumor antigen, a viral antigen, a bacterial antigen, afungal antigen, or a parasite antigen.
 145. The human cell of claim 142,wherein the human cell is a hematopoietic stem cell (HSC).
 146. Thehuman cell of claim 142, wherein the human cell is a myeloid progenitorcell or a lymphoid progenitor cell.
 147. The human cell of claim 142,wherein the human cell is a red blood cell.
 148. The human cell of claim142, wherein the human cell is selected from the group consisting of alymphocyte, a monocyte, a dendritic cell, a macrophage, a neutrophil, amast cell, an eosinophil, a basophil, and a natural killer (NK) cell.149. The human cell of claim 142, wherein the costimulatory molecule isa molecule that binds to a CD28 family receptor, a CD2 family receptor,ICOS, CD27, or 4-1BBL.
 150. The human cell of claim 142, wherein theagent comprises a costimulatory molecule, and the costimulatory moleculeis a B7 molecule, an ICOS ligand, a TNF alpha family member 4-1BBL,OX40, or OX40L.
 151. The human cell of claim 142, wherein the agentcomprises a costimulatory molecule, and the costimulatory molecule is4-1BBL.
 152. The human cell of claim 142, wherein the agent comprises anadjuvant, and wherein the adjuvant comprises a CD40 ligand, anti-CD40antibody, a ligand of a TLR, a pathogen-derived molecular pattern(PAMP), a PAMP mimic, an immunostimulatory nucleic acid, or a cationicpolymer.
 153. The human cell of claim 152, wherein the adjuvantcomprises a ligand of a TLR, and wherein the TLR is TLR3, TLR4, and/orTLR9.
 154. The human cell of claim 152, wherein the adjuvant comprises acationic polymer, and wherein the cationic polymer is a poly(aminoacid).
 155. The human cell of claim 142, wherein the agent comprises acytokine, and wherein the cytokine is an interleukin, TNF-alpha, acolony stimulating factor, interferon alpha 2a, interferon alpha 2b,interferon beta-1a, interferon beta-1b, leukemia inhibitory factor(LIF), or oncostatin M.
 156. The human cell of claim 155, wherein thecytokine is an interleukin, and wherein the interleukin is IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,membrane-bound IL-15, IL-15, IL-17, IL-21, IL-23, IL-27, IL-35, orIL-38.
 157. The human cell of claim 155, wherein the cytokine is aninterleukin, and wherein the interleukin is IL-15 or membrane-boundIL-15.
 158. The human cell of claim 155, wherein the cytokine is acolony stimulating factor, and wherein the colony stimulating factor isgranulocyte colony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), or macrophage colony-stimulatingfactor (M-CSF).
 159. The human cell of claim 142, wherein the sortaserecognition sequence comprises LPXTG.
 160. A method of modulating animmune response of a subject to an entity of interest, the methodcomprising administering to the subject the human cell of claim 142.161. A method of treating a subject in need of treatment for a disease,the method comprising administering to the subject the human cell ofclaim
 142. 162. The method of claim 161, wherein the disease comprisesan infectious disease, cancer, an autoimmune disease, an allergy, aninflammatory condition, or an immunodeficiency.
 163. A method ofconjugating an agent to a human cell, the method comprising contactingthe human cell with a sortase substrate that comprises both a sortaserecognition sequence and an agent, wherein the contacting is performedin the presence of a sortase under conditions suitable for the sortaseto conjugate the sortase substrate to an endogenous, non-engineeredpolypeptide of the human cell, wherein the agent comprises a cytokine,an antigen, a costimulatory molecule, or an adjuvant.